Assay buffer, compositions containing the same, and methods of using the same

ABSTRACT

The invention relates to improved electrochemiluminescence assay methods for phosphorylated peptides or proteins employing phospho-specific antibodies and buffer compositions that are substantially free of inorganic phosphate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application U.S. Ser. No.11/055,472, filed Feb. 9, 2005, which is a divisional of applicationU.S. Ser. No. 10/238,437, filed Sep. 10, 2002, which claims priority toU.S. Provisional Application Ser. No. 60/318,289, filed Sep. 10, 2001,and U.S. Provisional Application Ser. No. 60/363,498, filed Mar. 11,2002, each of which are hereby incorporated by reference.

1. FIELD OF THE INVENTION

This application relates to compositions for use in assays, particularlyin electrochemiluminescent assays, and methods of using the same.

2. BACKGROUND OF THE INVENTION

At this time, there are a number of commercially available instrumentsthat utilize electrochemiluminescence (ECL) for analytical measurementsincluding drug screening. Species that can be induced to emit ECL(ECL-active species) have been used as ECL labels. Examples of ECLlabels include: i) organometallic compounds where the metal is from, forexample, the noble metals of group VIII, including Ru-containing andOs-containing organometallic compounds such as thetris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and relatedcompounds. Species that participate with the ECL label in the ECLprocess are referred to herein as ECL coreactants. Commonly usedcoreactants include tertiary amines (e.g., see U.S. Pat. No. 5,846,485,herein incorporated by reference), oxalate, and persulfate for ECL fromRuBpy and hydrogen peroxide for ECL from luminol (see, e.g., U.S. Pat.No. 5,240,863). The light generated by ECL labels can be used as areporter signal in diagnostic procedures (Bard et al., U.S. Pat. No.5,238,808). For instance, an ECL label can be covalently coupled to abinding agent such as an antibody, nucleic acid probe, receptor orligand; the participation of the binding reagent in a bindinginteraction can be monitored by measuring ECL emitted from the ECLlabel. Alternatively, the ECL signal from an ECL-active compound may beindicative of the chemical environment (see, e.g., U.S. Pat. No.5,641,623 which describes ECL assays that monitor the formation ordestruction of ECL coreactants). For more background on ECL, ECL labels,ECL assays and instrumentation for conducting ECL assays see U.S. Pat.Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623;5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141;5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863;6,207,369 and 5,589,136 and Published PCT Nos. WO99/63347; WO00/03233;WO99/58962; WO99/32662; WO99/14599; WO98/12539; WO97/36931 andWO98/57154.

Commercially available ECL instruments have demonstrated exceptionalperformance. They have become widely used for reasons including theirexcellent sensitivity, dynamic range, precision, and tolerance ofcomplex sample matrices. The commercially available instrumentation usesflow cell-based designs with permanent reusable flow cells. Recently,ECL instrumentation has been disclosed that uses reagents immobilized onthe electrode used to induce ECL (see, e.g., U.S. Pat. Nos. 6,140,045;6,066,448; 6,090,545; 6,207,369 and Published PCT Application No.WO98/12539). Multi-well plates having integrated electrodes suitable forsuch ECL measurements have also been recently disclosed (see, e.g., U.S.application Ser. No. 10/185,274 (now U.S. Pat. No. 7,842,246) and Ser.No. 10/185,363 (now U.S. Pat. No. 6,977,722), each filed on Jun. 28,2002 and hereby incorporated by reference). See also, U.S. applicationSer. No. 10/238,391, filed on Sep. 10, 2002, now U.S. Pat. No.7,858,321, which is hereby incorporated by reference.

Currently, pH buffers containing inorganic phosphate are employed inmany electrochemiluminescence assays. Applicants have discovered thatsuch pH buffers can, in certain assays, interfere with the assay anddecrease the performance of the assay.

Accordingly, it would be desirable to find alternative pH assay buffers,compositions containing the same and methods of using the same for usein those assays which are detrimentally effected by pH bufferscontaining inorganic phosphate. It would also be desirable to findalternative ECL Assay Buffers with improved performance in ECL assays.

3. SUMMARY OF THE INVENTION

The present invention relates to improved compositions, reagents, kits,systems, system components, and methods for performing assays. Moreparticularly, the invention relates to the use of novel combinations ofreagents to provide improved assay performance.

One aspect of the invention relates to improved ECL Assay Buffers thatcomprise an ECL coreactant and, preferably, a pH buffering agent. TheECL Assay Buffers provide a suitable environment for efficientlyinducing ECL labels to emit ECL and for sensitively measuring ECL labelsvia the measurement of ECL. The ECL Assay Buffers of the invention mayoptionally comprise additional components including detergents,preservatives, anti-foaming agents, ECL active species, salts, metalions and/or metal chelating agents. The ECL Assay Buffers of theinvention may also include components of a biological assay, which insome cases may be labeled with an ECL label, including binding reagents,enzymes, enzyme substrates, cofactors and/or enzyme inhibitors. Theinvention also includes assay reagents, compositions, kits, systems andsystem components that comprise the ECL Assay Buffers of the inventionand, optionally, additional assay components. The invention alsoincludes methods for conducting ECL assays using the ECL Assay Buffersof the invention.

Another aspect of the invention relates to the use of pH buffers whichare substantially free of inorganic phosphates. Such buffers, in someapplications, have been found to significantly improve the performanceof ECL measurements. Such buffers have also been found to beadvantageous in certain applications where phosphate has been found tointerfere with a chemical, biochemical or biological reaction.

Surprisingly, such reagents provide a number of surprising advantagesincluding improving the performance of assays employing phospho-specificantibodies (i.e., antibodies that specifically bind with aphospho-peptide, phospho-amino acid and/or phospho-protein). It isbelieved that these antibodies may have a low affinity for inorganicphosphate and that the elimination of the inorganic phosphate greatlyreduces interference between the phosphate of the pH buffer and thephospho-specific antibodies. Accordingly, the invention includes method,reagents, kits and compositions for measuring phospho-peptides,phospho-amino acids or phospho-protein which use buffer compositionsthat are free or substantially free (e.g., below the levels thatinterfere with phospho-specific antibodies). Such methods, kits,compositions, and reagents are, preferably, applied to the measurement(most preferably using ECL detection) of protein kinase or phosphorylaseactivities through the specific measurement of reaction products orsubstrates.

Another aspect of the invention relates to compositions and reagentswith that give high signal to background ratios inelectrochemiluminescence assays. Such improved performance has beenachieved through the identification of advantageous combinations of ECLcoreactants, pH buffers, detergent and pH and, in particular, throughthe use of ECL coreactants and/or pH buffers other than TPA andphosphate. These improved formulations are of particular value innon-wash assays and high sensitivity assays. In some embodiments of theinvention, the performance of ECL assays is improved even furtherthrough optimal combinations of reagent compositions with electrodecompositions.

In some embodiments of the invention, the compositions and reagents ofthe invention improve the ratio of ECL signal from bound label to ECLsignal from free label. This is particularly true in assays involvingreagents immobilized on a solid surface such as an electrode. This isimportant, for example, in solid phase assays not having a wash step(especially in low affinity interaction assays) since the majorcomponent of the background signal comes from the labels present insolution.

Yet another advantage of the invention relates to improved sensitivityof assays using the compositions of the invention. More specifically,the ECL Assay Buffers of the invention provide improved sensitivity atlow detection levels by reducing the background electrochemiluminescencein the absence of ECL labels. Surprisingly, ECL Assay Buffers comprisingpH buffering agents other than phosphate or which are substantially freeof inorganic phosphate emit less background luminescence thanconventional ECL Assay Buffers comprising inorganic phosphate based pHbuffers. This is particularly advantages at low detection levels whereincreasing the signal to background ratio greatly improves theperformance of the assay.

Another aspect of the invention relates to improved reagent kitscomprising the ECL assay buffers, where the reagents includenon-phosphate based pH buffering agents, the ECL assay buffers aresubstantially free of inorganic phosphate and/or the ECL assay buffersemploy tertiary amine coreactants other than TPA. In particular, kitscontaining, in one or more containers, the ECL assay buffer and,preferably also containing one or more other assay components.

Another aspect of the invention relates to improved methods performedusing the present invention, particularly assay methods employingphospho-specific antibodies, low detection limits, immobilized reagentsand/or a non-wash formats.

Yet another aspect of the invention relates to improved systems andapparatus containing the compositions or reagents of the inventionand/or improved systems and apparatus adapted to perform the improvedmethods of the invention.

4. DESCRIPTION OF THE FIGURES

FIG. 1 compares the rates of dissociation of a phosphopeptideantiphosphopeptide complex in three different ECL Assay Buffers thatcomprise different pH buffering agents.

FIG. 2 shows the rate of dissociation of aphosphopeptide-antiphosphopeptide complex in an ECL Assay Buffer thatcomprises TPA as an ECL coreactant and Tris as a pH buffering agent. Thecomplex was not washed to remove free antibody prior to addition of theECL Assay Buffer.

FIG. 3 is a graphical representation of an end-product stability studycomparing the dissociation rate of an anti-phosphotyrosine antibody fromautophosphorylated EGF receptor in two different ECL Assay Buffers: 150mM TPA/150 mM Phosphate and 100 mM TPA/400 mM glycyl-glycine. Theconcentration of the labeled α-phosphotyrosine antibody was 6.7 nM.

FIG. 4 compares the performance of four different ECL Assay Buffers inthe ECL measurement of a labeled reagent that was immobilized on thesurface of an unetched (FIG. 4A) or plasma etched (FIG. 4B) carbon inkelectrode. The figure shows the signals from surface bound reagent, thebackground signal measured in the absence of the bound reagent and thesignal to background ratio (S/B).

FIG. 5 compares the performance of four different ECL Assay Buffers inthe ECL measurement of a labeled reagent that was immobilized on thesurface of an unetched (FIG. 5A) or plasma etched (FIG. 5B) carbon inkelectrode. The figure shows the signals from surface bound reagent, thebackground signal measured in the absence of the bound reagent and thesignal to background ratio (S/B). The figure also shows the signalobtained when a non-surface bound labeled reagent was introduced intothe ECL Assay Buffers and the ratio of the signals from the surfacebound and non-surface bound reagents (B/F).

FIG. 6 compares the effect of three different detergents on the ECLsignal from a labeled reagent that was immobilized on the surface of aplasma etched carbon ink electrode. The detergents were introduced intoan ECL Assay Buffer comprising TPA and phosphate (FIG. 6A) or PIPES andphosphate (FIG. 6B). The figure shows the signals from surface boundreagent, the background signal measured in the absence of the boundreagent and the signal to background ratio (S/B).

FIG. 7 compares the effect of five different detergents on the ECLsignal from a labeled reagent that was immobilized on the surface of anon-etched carbon ink electrode. The detergents were introduced intofour different ECL Assay Buffers differing in the identity of the BCLcoreactant or pH buffering agent. The figure shows the signals fromsurface bound reagent, the background signal measured in the absence ofthe bound reagent and the signal to background ratio (S/B).

5. DETAILED DESCRIPTION OF THE INVENTION

The invention, as well as additional objects, features and advantagesthereof, will be understood more fully from the following detaileddescription of certain preferred embodiments.

An ECL-active species may be referred to as an ECL moiety, ECL label,ECL label compound or ECL label substance, etc. It is within the scopeof the invention for these ECL-active species—when utilized in certainof the composition, reagent, kit, method, or system embodiments inaccordance with the invention—to be linked to other molecules and, inparticular, to components of biochemical or biological assays, e.g., ananalyte or an analog thereof, a binding partner of the analyte or ananalog thereof, a further binding partner of such aforementioned bindingpartner, or a reactive component capable of binding with the analyte, ananalog thereof or a binding partner as mentioned above. Theabove-mentioned species can also be linked to a combination of one ormore binding partners and/or one or more reactive components. In certainenzymatic assays, an ECL-active species may be linked to an enzymesubstrate.

It is similarly within the scope of the invention for the aforementioned“composition”, hereinafter sometimes an “ECL, composition”, or a“system” to contain unstable, metastable and other intermediate speciesformed in the course of the ECL reaction, such as an ECL moiety in anexcited state as aforesaid and the above-mentioned strong reducingagent. Additionally, although the emission of visible light is anadvantageous feature of certain embodiments of the invention it iswithin the scope of the invention for the composition (hereinaftersometimes “ECL composition”) or system to emit other types ofelectromagnetic radiation, such as infrared or ultraviolet light,X-rays, microwaves, etc. Use of the terms “electrochemiluminescence”,“electrochemiluminescent”, “electrochemiluminesce”, “luminescence”,“luminescent” and “luminesce” in connection with the present inventiondoes not require that the emission be light, but admits of theemission's being such other forms of electromagnetic radiation.

The present invention relates to ECL assay buffers, assay compositionscontaining the same, and methods of using the same. As stated above,several disadvantages were discovered when using the phosphate based ECLassay buffer of the prior art. More specifically, it was found duringthe development of a specific ECL assay for tyrosine kinase activitythat the phosphate in a standard formulation of the ECL coreactant TPA(ORIGEN Assay Buffer, IGEN International: 200 mM Phosphate, ˜100 mM TPA,pH˜7.5) disrupted the binding between an phospho-specific antibody and aphosphorylated substrate. The assay involved i) the kinase-dependentphosphorylation of a peptide immobilized on a carbon electrode; ii) thespecific binding of a labeled (with a derivative of Ru(bpy)₃)phospho-specific antibody; iii) the addition of the ECL coreactanttripropylamine (TPA) and iv) the detection of ECL from the bound label(see, e.g., Example A below). Applicants discovered that when TPA wasadded by the addition of ORIGEN Assay Buffer that the measured ECLsignal was sharply dependent on the time the binding complex was leftexposed to the ORIGEN Assay Buffer (FIG. 1); the measured signal droppedsharply over time. In fact, after a 1-hour incubation only a smallfraction (˜10%) of initial signal was detected. The affinity of the pY20antibody (Zymed Lab) used in the assay toward the phospho-tyrosine sitesthat were formed at the surface of the plate during the enzymaticreaction was much greater than toward free phosphate in solution.However, the high concentration of free phosphate in ORIGEN assay buffer(200 mM) is now believed to have caused the dissociation ofphospho-tyrosine/pY20 complex, resulting in the signal decaying sharply.

One way around this problem was to have a fixed time between thedispensing of ECL assay buffer and the read step so that the signaldecay is calibrated and subtracted. However, this approach is notdesirable in high throughput screening applications, where robustness ofthe assay and flexibility of dispensing protocol are desired.

Thus, a number of different organic pH buffers were tested asalternatives to the conventional phosphate based assay buffer. Many ofthe conventional biological buffers (including Tricine, HEPES, MOPS,BES—all from Sigma), however, interfered with the ECL generation fromTPA and provided only 2-20% of ECL signal observed with the ORIGEN assaybuffer. Applicants, however, discovered a set of buffers that providedECL signals that were comparable to the signal observed inTPA/phosphate.

Accordingly, applicants have discovered that substitution of thephosphate buffer with a pH buffer which was substantially free ofinorganic phosphate can ECL signal comparable to the signal observed instandard ORIGEN assay buffer, without the above-described disadvantages.Preferably, the pH buffer is free of inorganic buffer.

Furthermore, applicants have discovered that the phosphate-free ECLassay buffers of the invention are not only beneficial when applied tophosphopeptide binding assays but have other beneficial properties(including lower background signals) that may improve a wide range ofECL assays.

Furthermore, applicants have discovered ECL assay buffer backgroundreducing agents that, when introduced into ECL assay buffers reduce ECLassay buffer background and improve assay performance. These agents are,preferably, also pH buffering agents, most preferably, GlyGly or Tris.

Furthermore, applicants have discovered novel ECL assay buffers thatemploy ECL coreactants other than the traditional TPA. Surprisingly, anumber of coreactants have been discovered to generate ECL signals thatare comparable to those generated with TPA. In addition, the use of ECLcoreactants other than TPA have been found to improve the performance ofnon-washed ECL assays through their improved ability, relative to TPA,to discriminate between ECL labels that are held in proximity to anelectrode and labels that are free in solution. The use of coreactantsother than TPA has additional benefits due to the higher watersolubility and lower vapor pressure of some of the new coreactants thathave been identified.

Furthermore, applicants have discovered that the presence or absence ofdetergents can have profound impact on the performance of an ECL assaybuffer. Surprisingly, the effect of detergents on ECL can be influencedby the choice of ECL coreactant and working electrode material.Applicants have developed detergent-containing ECL assay bufferssuitable for a variety of different applications and ECL systems.

As noted above, one aspect of the invention relates to improved ECLAssay Buffers that comprise an ECL coreactant and, preferably, a pHbuffering agent. The ECL Assay Buffers provide a suitable environmentfor efficiently inducing ECL labels to emit ECL and for sensitivelymeasuring ECL labels via the measurement of ECL. The ECL Assay Buffersof the invention may optionally comprise additional components includingdetergents, preservatives, anti-foaming agents, ECL active species,salts, metal ions and/or metal chelating agents. The ECL Assay Buffersof the invention may also include components of a biological assay,which in some cases may be labeled with an ECL label, including bindingreagents, enzymes, enzyme substrates, cofactors and/or enzymeinhibitors.

Preferably, the ECL assay buffers of the invention are aqueous orsubstantially aqueous in nature, although it may be desirable in someapplications to add organic cosolvents such as DMSO, DMF, methanol,ethanol or other alcohols. In one embodiment of the invention, an ECLassay buffer (or one or more components thereof) is provided in dry formand the user forms the ECL assay buffer solution by addition of theappropriate solvent or matrix (preferably a water or an aqueous medium).

5.1 ECL Coreactants

Most, if not all, current commercial applications of ECL technologyinvolve the measurement of ECL labels (and, in particular,organometallic complexes of ruthenium) in the presence of an ECL assaybuffer containing tri-n-propylamine (TPA) as a coreactant and phosphateas a pH buffering agent. These ECL assay buffers have been optimized forand have provided excellent performance in commercial ECLinstrumentation that employ, as a solid phase for binding assays,magnetic particles that are collected on the surface of a metal(typically, platinum) electrode.

Applicants have discovered that in some applications, certainfunctionalized tertiary alkylamines can provide performance that iscomparable or better to TPA. These functionalized tertiary amines areespecially useful in assays employing carbon-based electrodes (e.g.,electrodes comprising carbon particle or carbon nanotubes includingcomposite materials such as plastics and inks) and/or assay reagents(such as binding reagents) that are immobilized onto electrodes. Thefunctionalized tertiary alkylamines of the invention, preferably, haveone or more of the following properties: i) they are oxidized oncarbon-based electrodes in a one electrode oxidation to give an amineradical cation which can subsequently lose a proton to form a radicalreductant (Scheme 1); ii) they have an oxidation potential oncarbon-based electrodes that is comparable (within 150 mV) or greaterthan that of Ru(II)(bpy)₃; iii) they can be oxidized, most preferably ata pH between 6 and 9, at a potential less than that required tobreakdown water at a carbon-based electrode; iv) the energy released bythe reaction of the radical reductant with Ru(III)(bpy)3 to produceRu(II) bpy)3 is sufficient to produce Ru(II)(bpy)3 in a luminescentexcited state and v) the lifetimes of the amine radical cation and/orradical reductant are less than the corresponding TPA-derived species.

Applicants have discovered that, through the use of the functionalizedtertiary alkylamines of the invention, it is possible to improve theselectivity of ECL excitation at an electrode for ECL labels bound tothe electrode (as opposed to ECL labels that are free in solution).Without being bound by theory, it is believed that this increasedselectivity is due to the lower lifetimes of the amine radical cationand/or radical reductant relative to the corresponding TPA-derivedspecies (thus limiting the participation of the reactive species to ECLreactions that occur proximate to the electrode surface). Preferably,the diffusion distance of the amine radical cation and/or radicalreductant (the distance that the species can diffuse during itslifetime) is less than 1 μm, more preferably, <500 nm, even morepreferably less than 100 nm, even more preferably less than 50 nm andmost preferably <10 nm. The high selectivity between free and boundlabels has led to improved sensitivity in non-washed ECL assay formats.The ratio of signal from bound label and free label (B/F ratio) mayimproved by replacing TPA with a non-TPA coreactant of the invention.This improvement is preferably greater than a factor of 2, morepreferably greater than a factor of 5 and most preferably greater than afactor of 10.

The functionalized tertiary amine coreactants of the invention,preferably have the structure NR¹R²R³, wherein R¹, R² and R³ are alkylgroups comprising at least 2, preferably 3, carbons and wherein one ormore of R, R² and R³ are functionalized with a hydrophilic functionalgroup, more preferably a charged group, most preferably a negativelycharge group. Preferred functional groups include hydroxyl,dialkylamino, sulfate, sulfonate, carboxylate and carboxylic acid ester.

Especially preferred coreactants include compounds with the structure(n-Pr)₂N(CH₂)_(n)R, wherein n is greater than or equal to 2 (morepreferably 3), and R is a hydrophilic functional group as defined above,preferably, carboxylate, dialkylamino (more preferably dipropylamino) ormost preferably sulfonate.

Other preferred coreactants include compounds with the structure

Wherein i) X is —(CH₂)— or a heteroatom, preferably —O—, —S—, or—N(R¹)—; ii) R and R¹ are alkyl groups comprising 2 or more (preferably3 or more) carbons; iii) n and m are each greater than or equal to 1 andare preferably two and iv) R (and, optionally R¹) comprise a hydrophilicfunctional group as defined above. Most preferably, R is —(CH₂)_(n)—F¹,wherein n is greater than or equal to 3 and F¹ is a hydrophilicfunctional group, preferably, carboxylate or sulfonate. In the caseswhere X is N(R¹)—, R¹ is, most preferably, —(CH₂)_(n)—F², wherein n isgreater than or equal to 3 and F¹ is H, alkyl, or a hydrophilicfunctional group, most preferably, carboxylate or sulfonate.

Many of the so-called “Good” buffers (Good et al., Biochemistry, 5, 467(1966); Good et al., Methods in Enzymol., 24, Part B, 53 (1972) andFerguson et al., Anal. Biochem., 104, 300 (1980)) have tertiary aminesand have been found to act as ECL coreactants on carbon electrodes.These “Good” buffers, generally have tertiary amines having piperazineor morpholine cores. Specific amines that act as ECL coreactants oncarbon-based electrodes include: 3-(di-n-propylamino)-propanesulfonicacid; 4-(di-n-propylamino)-butanesulfonic acid;4-[bis-(2-hydroxyetane)-amino]-butanesulfonic acid;piperidine-N-(3-propanesulfonic acid); azepane-N-(3-propanesulfonicacid); piperidine-N-(3-propionic acid) (PPA);3-morpholino-2-hydroxypropanesulfonic acid (MOPSO);3-morpholinepropanesulfonic acid (MOPS);N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (EPPS);N-(2-hydroxyethyl)piperazine-N′-3-ethanesulfonic acid (BES);piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES); triethanolainine;N-2-hydroxypiperazine-N-2-ethanesulfonic acid (HEPES);piperazine-N,N′-bis-4-butanesulfonic acid;homopiperidine-N-3-propanesulfonic acid;piperazine-N,N′-bis-3-propanesulfonic acid;piperidine-N-3-propanesulfonic acid;piperazine-N-2-hydroxyethane-N′-3-methylpropanoate;piperazine-N,N′-bis-3-methylpropanoate;1,6-diaminohexane-N,N,N′,N′-tetraacetic acid; N,N-bispropyl-N-4-aminobutanesulfonic acid;N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES);1,3-bis[tris(hydroxymethyl)methylamino]propane (bis-Tris propane);3-dimethylamino-1-propanol; 3-dimethylamino-2-propanol;N,N,N′,N′-tetrapropylpropane-1,3,-diamine (TPA dimer);piperazine-N,N′-bis(2-hydroxypropane)sulfonic acid (POPSO) and2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid(HEPPSO). HEPES. POPSO, HEPPSO, EPPSO, PPA and PIPES are especiallypreferred for their high signals and high discrimination between boundand free labels. TES is also preferred for its high signal.

Additional coreactants include proline, peptides having an N-terminalproline. Preferably, the proline is N-alkylated to form a tertiaryamine.

The use of coreactants having hydrophilic functional groups (and, inparticular, coreactants that are zwitterionic at neutral pH) has avariety of advantages that are unrelated to their ability to act as ECLcoreactants. These species tend to be highly water soluble and to havelow vapor pressure. For these reasons it is possible to produce highlyconcentrated stock solutions that may be diluted as necessary for use.It is also possible prepare dried reagents comprising the coreactantswithout uncertainty due to loss of coreactant in the vapor phase.Furthermore, when present in dried reagents, these coreactantsresolubilize quickly in a minimum of volume.

5.2 pH Buffering Agents

Conventional ECL assay buffers optimized for use with commercial ECLinstruments have typically comprised TPA in a phosphate-based pH buffer.These formulations have been especially useful for conducting solidphase assays employing magnetic particles that are captured on anelectrode. Applicants have discovered that in some applications, otherpH buffering agents (including organic pH buffers) can provideperformance that is comparable or better to phosphate. Thesenon-phosphate pH buffers (and pH buffer solutions and ECL assay bufferscomprising these buffers are especially useful in assays employingcarbon-based electrodes (e.g., electrodes comprising carbon particle orcarbon nanotubes including composite materials such as plastics andinks) and/or assay reagents (such as binding reagents) that areimmobilized onto electrodes. They are also advantageous for use inassays where phosphate is an interferent. Preferably, ECL assay buffersemploying the non-phosphate buffers of the invention have less than 15mM inorganic phosphate, more preferably they have less than 5 mMinorganic phosphate, even more preferably they have less than 1 mMphosphate, even more preferably they are substantially free of inorganicphosphate, most preferably they are free of inorganic phosphate.

The pH buffering agent, preferably, is not oxidized under the conditionsused to generate ECL and do not interfere with the generation of ECL.Two pH buffers that have proved especially useful aretris-(hydroxymethyl)aminomethane (Tris) and oligo(glycines), preferablyglycyl-glycine (Gly-Gly). Applicants have discovered that ECL assays oncarbon-based electrodes using TPA/Tris or TPA/Gly-Gly ECL assay buffershave comparable signals from electrode-bound ECL labels as thoseobserved with conventional TPA/phosphate buffers. The background signalsin the absence of ECL labels, however, are considerably less with theTris and Gly-Gly buffers. This reduction in the background signal leadsto an increase in the ratio of signal to background (S/B) and anincrease in the sensitivity of ECL assays using the new formulations.Preferably, the ECL assay buffers of the invention have S/B ratios thatare 2-fold, more preferably 5-fold and, most preferably, 10-fold betterthan those obtained using phosphate-based systems.

Without being bound by theory, applicants hypothesize that the improvedperformance of the Tris and Gly-gly based ECL assay buffers is relatedto an ability of the buffering agents to act as ECL assay bufferreducing agents by reacting with and destroying tertiary amine oxidationproducts and/or other reactive oxidized species (e.g., amine radicalcations and radical reductants) that are responsible for the assaybuffer background. This effect is most pronounced away from theelectrode surface where the concentration of these species are lower, sothe Tris and Gly-gly components do not affect signals fromelectrode-bound labels. The Tris and Gly-gly buffers may also improvethe observed bound to free ratios, although this effect is less thanthat observed by switching to non-TPA buffers such as PIPES.

Applicants have found that the Tris and Gly-gly buffering systems arealso suitable for use with non-TPA coreactants such as PIPES. When usingcoreactants such as PIPES that may act as pH buffers, it may be possibleto omit additional buffering agents.

5.3 Detergent

Applicants have discovered that the presence or absence of detergentscan have a surprisingly large effect on ECL signals. The nature of thiseffect is, unexpectedly, dependent on the electrode. On oxidizedelectrodes (e.g., plasma-oxidized carbon inks or plasma oxidized polymercomposites containing carbon particles or carbon nanotubes) exposed toTPA-containing ECL assay buffers, the effect appears to be relativelysmall except in the case of phenyl ether containing detergents such asthe Triton and Nonidet series of detergents (e.g., Triton X-100). Acommon method for generating ECL is through the use of a ramp potential.In general a plot of ECL intensity vs. applied potential has the form ofa peak. ECL increases as the oxidation potentials of the label andcoreactant are approached. On scanning past this potential, the ECLintensity eventually begins to drop as the coreactant is consumed andwater oxidation begins to occur. Applicants have observed that theaddition of phenyl ether containing detergents leads to the addition ofa small ECL peak at higher potential than the main ECL peak. This peakoccurs at a potential similar to the an oxidation wave observed withpure Triton X-100, thus leading applicants to speculate that the newpeak is associated with the oxidation of the detergent (or an associatedimpurity) and the participation of the oxidation products in an ECLreaction.

The behavior on non-oxidized carbon-based electrodes (and, inparticular, untreated carbon ink electrodes) is very different. On theseelectrodes the ECL signal in the presence of TPA-containing buffers (aswell as the S/B ratio) is drastically improved by the addition ofdetergent. This effect appears to be relatively independent of thenature of the detergent (although non-ionic detergents are preferred dueto their relatively weak ability to denature biological systems), butrequires the concentration of detergent to be roughly equal to orgreater than the critical micellar concentration (cmc) of the detergent.In preferred embodiments of the invention, the addition of detergent toan ECL assay buffer leads to an improvement in assay signal or S/B(preferably induced with a carbon-based electrode, most preferably acarbon-ink electrode) of greater than a factor of 2, more preferablygreater than a factor of 5 and most preferably greater than a factor of10.

The behavior of non-TPA containing ECL assay buffers and, in particular,non-TPA containing ECL assay buffers (especially, buffers comprising thenon-TPA tertiary amine coreactants of the invention, preferablycomprising N-substituted morpholines or piperazines, most preferablycomprising PIPES) appears to be less dependent on the nature of a carbonelectrode. For example, applicants have found that assays involving theuse of PIPES as a coreactant, on both oxidized and non-oxidizedelectrodes, are unexpectedly and significantly improved by the additionof phenyl ether containing substances, and, in particular, phenyl ethercontaining detergents. Other detergents that did not possess the phenylether moiety did not produce this effect. In preferred embodiments ofthe invention, the addition of detergent to a non-TPA based ECL assaybuffer (preferably, a PIPES-based ECL assay buffer) leads to animprovement in assay signal or S/B (preferably induced with acarbon-based electrode, most preferably a carbon-ink electrode) ofgreater than a factor of 10, more preferably greater than a factor of 30and most preferably greater than a factor of 100.

In certain assays, e.g., assays involving detergent sensitive componentssuch as biological membranes, it may be advantageous to reduce (e.g., to<0.1%) or eliminate detergents from ECL Assay Buffers. It should beunderstood that the various detergent containing formulations of theinvention may also be prepared in low detergent or detergent-free formsfor these detergent sensitive applications. In preferred embodiments,assays employing detergent sensitive components employ ECL Assay Bufferscontaining one of the following coreactants; TPA,N,N-bis-(hydroxyethyl)-N-4-aminobutanesulfonic acid, orA₂N—(CH₂)_(n)—NB₂, where A and B are alkyl groups (preferably, propyl)and n is an integer (preferably 3 or 4, most preferably, 3).

5.4 Preservatives

It may be beneficial when storing ECL assay buffers to include apreservative that prevents microbial growth. Preferably, thepreservative has little or no effect on ECL generated using the ECLassay buffer, especially when using the ECL assay buffer on a carbonbased electrode. Azide has been found to be a suitable preservative.Isothiazolones (e.g., Kathon, 2-methyl-4-isothiazolin-3-one and5-chloro-2-methyl-4-isothiazolin-3-one), oxazolidines (e.g., Oxaban A or4,4 dimethyl oxazolidine) and related preservatives are especiallypreferred due their compatibility with ECL, their high activity and thelow degree of problems associated with safety hazards or environmentalconcerns.

5.5 Anti-Foam Agents

It may be beneficial, especially in HTS applications, to avoid theproduction of bubbles or foam. For this reason it may be desirable toadd anti-foaming agents to ECL assay buffers. Applicants have found thatmany commercial anti-foaming agents (including Antifoams o-30, Antifoam204, Antifoam A, Antifoam SE-15, Antifoam SO-25 and Antifoam 289) may beadded to ECL assay buffers without significantly affecting theperformance of the ECL assay buffers.

5.6 ECL Labels

The compositions of the invention may include ECL labels. The ECL labelsmay be conventional ECL labels. Examples of ECL labels include: i)organometallic compounds where the metal is selected from, for example,the noble metals of group VIII, including Ru-containing andOs-containing organometallic compounds such as thetris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and relatedcompounds. Preferably, the ECL labels are capable of repeatedly emittingelectrochemiluminescence. Preferred ECL labels are ruthenium orosmium-containing organometallic species. More preferably, theseruthenium or osmium-containing organometallic comprise ruthenium orosmium chelated to polypyridyl ligands (most preferably, bipyridine,phenanthroline, and/or substituted derivatives thereof). Mostpreferably, the ECL labels comprise ruthenium-tris-bipyridine, thebipyridine ligands being, optionally substituted, e.g., with a linkinggroup for attaching the label to an assay reagent.

The ECL label may be linked to an assay reagent, optionally through alinking group. Examples of binding reagents that may be linked to an ECLlabel include: whole cells, cell surface antigens, subcellular particles(e.g., organelles or membrane fragments), viruses, prions, dust mites orfragments thereof, viroids, antibodies, antigens, haptens, fatty acids,nucleic acids (and synthetic analogs), proteins (and synthetic analogs),lipoproteins, polysaccharides, inhibitors, cofactors, haptens, cellreceptors, receptor ligands, lipopolysaccharides, glycoproteins,peptides, polypeptides, enzymes, enzyme substrates, enzyme products,second messengers, cellular metabolites, hormones, pharmacologicalagents, synthetic organic molecules, organometallic molecules,tranquilizers, barbiturates, alkaloids, steroids, vitamins, amino acids,sugars, lectins, recombinant or derived proteins, biotin, avidin,streptavidin. The assay reagents are preferably useful as bindingreagents or enzyme substrates in, e.g., binding assays or enzyme assays.

5.7 Compositions

One aspect of the invention relates to compositions comprising the ECLassay buffers of the invention.

Another aspect of the present invention relates to compositions suitablefor use in an assay comprising a pH buffer substantially free ofinorganic phosphate. Suitable pH buffers include glycylglycine(“Glygly”), tris(hydroxymethyl)aminomethane (“Tris”) or combinationsthereof. Other pH buffers which are also substantially free of or do notcontain inorganic phosphate would also be suitable for use in theinvention.

According to one embodiment of the invention, the composition comprisesa pH buffer, wherein the composition is, preferably, substantially freeof inorganic phosphate and, preferably further comprises one or more ECLco-reactants (preferably, TPA or alternatively, a non-TPA coreactant,more preferably an N-substituted morpholine or piperazine, mostpreferably PIPES). According to a particularly preferred embodiment, thecomposition is free of inorganic phosphate. Suitable pH buffers includeglygly and tris. Additional buffers may be selected on the basis ofcertain preferred characteristics: i) the ability to buffer in the pHrange of 6.5-8.5, preferably 7-8 (more preferably, the pKa of the bufferis in the range of 6.5 to 8.5 or more preferably, from 7.5 to 8.5); ii)commercial availability at low cost; iii) the lack of an inhibitoryeffect on ECL and/or iv) the lack of a significant oxidation wave in therange of 0-1.2 V or more preferably 0-1.5 V (the voltage window for theoxidation of Ru(bpy)₃ and TPA).

According to another embodiment of the invention, the compositioncomprises a non-phosphate pH buffering agent and, preferably furthercomprises one or more ECL co-reactants (preferably, TPA oralternatively, a non-TPA coreactant, more preferably an N-substitutedmorpholine or piperazine, most preferably PIPES). Preferably, thecomposition has less than 15 mM inorganic phosphate, more preferably ithas less than 5 mM inorganic phosphate, even more preferably it has lessthan 1 mM phosphate, even more preferably it is substantially free ofinorganic phosphate, most preferably it is free of inorganic phosphate.Suitable pH buffers include glygly and tris. Additional buffers may beselected on the basis of certain preferred characteristics: i) theability to buffer in the pH range of 6.5-8.5, preferably 7-8 (morepreferably, the pKa of the buffer is in the range of 6.5 to 8.5 or morepreferably, from 7.5 to 8.5); ii) commercial availability at low cost;iii) the lack of an inhibitory effect on ECL and/or iv) the lack of asignificant oxidation wave in the range of 0-1.2 V or more preferably0-1.5 V (the voltage window for the oxidation of Ru(bpy)₃ and TPA).

Preferably, the ECL co-reactant used in these embodiments is suitablefor use in an electrode induced luminescence reaction (e.g.,electrochemiluminescence). Suitable ECL co-reactants includetripropylamine (TPA). Non-TPA coreactants (preferably, tertiary aminesother than TPA as described in the coreactants section above) may beadvantageous in some applications, in particular, in non-washed assayformats.

Preferably, the composition comprises between 10 and 2000 mM pH buffer,more preferably 50 and 1200 mM, even more preferably between 100 and 600mM, and most preferably between 300 and 500 mM.

Preferably, the composition comprises between 10 and 1000 mM, ECLco-reactant, more preferably 30 and 600 mM, even more preferably between50 and 200 mM, and most preferably between 75 and 150 mM.

The optimal range of TPA concentrations in the pH buffers containingTris and Gly-Gly is very similar to concentrations of ORIGEN® buffer(i.e., ranging from 50-200 mM). The tested range of concentrations ofTris and Gly-Gly buffers is 100-600 mM. Preferably, the concentration is200 mM. ECL assay buffers comprising non-TPA coreactants of theinvention (preferably, PIPES) may include similar ranges of coreactantconcentrations, although in many applications the preferred range is10-100 mM, most preferably 20-50 mM.

According to another preferred embodiment, the final formulation of theGly-Gly/TPA buffer is: 200 mM Gly-Gly, 100 mM TPA, 0.1% Triton at pH7.8±0.05.

According to another preferred embodiment, the final formulation of theGly-Gly/TPA buffer is: 50-1000 mM Gly-Gly, 50-1000 mM TPA, at pH 7.8±1.Preferably, the formulation also comprises 0.2%-2% Triton X-100 and/or20-500 mM salt.

According to another preferred embodiment, the final formulation for theTris/TPA buffer is: 200 mM Tris, 100 mM TPA, 0.1% Triton at pH 7.8±0.05.

According to another preferred embodiment, the final formulation for theTris/TPA buffer is: 50-1000 mM Tris, 50-1000 mM TPA, at pH=7.8±0.05.Preferably, the formulation also comprises 0.2%-2% Triton X-100. and/or20-500 mM salt.

According to another preferred embodiment, the final formulation for thePIPES/Phos buffer is: 40-1000 mM phosphate (preferably, potassiumphosphate), 10-200 mM PIPES, at pH=7.8±0.05. Preferably, the formulationalso comprises 0.2%-2% Triton X-100.

Using Tris and Gly-Gly assay buffers significantly improved thestability of phosphopeptide-anti-phosphopeptide complexes in ECL-basedTyrosine Kinase assays. However, some dissociation of the complexes wasobserved in Tris buffer, although at much slower rates than in ORIGENassay buffer. In the case of the Gly-Gly buffer, ECL signal slowlyincreased, because no stop reagent was introduced into assay solution toquench the enzymatic reaction.

According to one preferred embodiment, the composition further comprisesa stop reagent (i.e., a reagent added to stop a reaction or reduceinterference with a reaction). Chelating agents such asethylenediaminetetraacetic acid (EDTA) are common stop reagents inMg-dependent kinase assays. EDTA binds Mg²⁺ ions that are require forsuccessful activation of ATP. The addition of 5 mM EDTA into Gly-Glyassay buffer, for example, helps to stop residual tyrosine kinaseenzymatic activity. Dissociation of phosphopeptide-anti-phosphopeptidecomplexes in Gly-Gly/TPA buffer with 5 mM EDTA does not exceed 1% per 1hour in a non-washed assay format. At concentrations higher than 10 mM,EDTA may have a negative effect on absolute value of ECL signal, butdoes not compromise stability of ECL signal upon incubation in assaybuffer. Depending on desired final read volume in 96-well plates (100 μlor 250 μl) and the type of assay (washed or non-washed) formulation ofGly-Gly/TPA solution may be different.

Preferably, the composition has a pH ranging from 6 to 9, morepreferably from 7 to 8, even more preferably from 7.5 to 8 and mostpreferably about 7.8. According to one preferred embodiment, the pH isadjusted by addition of an acid or base, preferably KOH, more preferably10% KOH.

One embodiment of the invention relates to an ECL assay buffercomprising:

(a) glycylglycine (Gly-Gly), preferably from 0.1 to 0.7 M, morepreferably 0.3 and 0.5 M, and most preferred about 0.2 M; and

(b) tripropylamine (TPA), preferably from 0.01 M to 0.3 M, morepreferably 0.05 to 0.2, and most preferred about 0.1 M.

Preferably, the assay buffer further comprises EDTA (preferably 1 to 10mM, more preferably 5 mM). Preferably, the assay buffer has a pH rangingfrom 6 to 9, more preferably from 7 to 8, even more preferably from 7.5to 8 and most preferably about 7.8. According to one preferredembodiment, the pH is adjusted by addition of an acid or base,preferably KOH, more preferably 10% KOH.

Another preferred embodiment of the invention relates to an ECL assaybuffer comprising:

(a) tris[hydroxymethyl)aminomethane (Tris), preferably from 0.1 to 0.7M, more preferably 0.3 to 0.5 M and most preferred about 0.2 M; and

(b) tripropylamine (TPA), preferably from 0.01 M to 0.3 M, morepreferably from 0.05 to 0.2 M and most preferred about 0.1 M.

Preferably, the assay buffer further comprisesethylenediaminetetraacetic acid (EDTA), preferably 1 to 10 mM, morepreferably 5 mM. Preferably, the composition has a pH ranging from 6 to9, more preferably from 7 to 8, even more preferably from 7.5 to 8 andmost preferably about 7.8. According to one preferred embodiment, the pHis adjusted by addition of an acid or base, preferably KOH, morepreferably 11% KOH.

Another preferred embodiment of the invention relates to ECL assaybuffers comprising coreactants other than TPA, preferably trialkylaminespresenting hydrophilic functional groups (as described in thecoreactants section). Preferably the coreactant is PPA or PIPES, mostpreferably PIPES. The concentration of coreactant is, preferably,between 10 and 800 mM, most preferably between 10 and 200 mM, mostpreferably between 20 and 50 mM. Preferably, the ECL assay buffer alsocomprises a pH buffering agent, preferably, phosphate, Tris or Gly-Gly.The concentration of the pH buffering agent is preferably between 0 and800 mM, more preferably between 0 and 400 mM, even more preferablybetween 20 and 200 mM and most preferably between 75 and 150 mM.Preferably, the composition has a pH ranging from 6 to 9, morepreferably from 7 to 8, even more preferably from 7.2 to 7.8 and mostpreferably about 7.5. Preferably, the ECL assay buffer also includes asubstance with a phenyl ether moiety and/or a detergent, preferably anon-ionic detergent, even more preferably a phenyl ether containingdetergent, most preferably Triton X-100. Preferably the concentration ofdetergent is greater than 0.02%, more preferably greater than 0.05%,most preferably between 0.05 and 0.5%.

According to one preferred embodiment, the reagents or compositions ofthe invention further comprise one or more detergents and/or surfactants(e.g., classes of non-ionic detergents/surfactants known by the tradenames of Nonidet, Brij, Triton, Tween, Thesit, Lubrol, Genapol,Pluronic, Tetronic, F108, and Span). Especially preferred detergentsinclude: Tween 20, Triton X-100, NP-40 and Thesit.

Another preferred embodiment of the invention relates to reagentcompositions comprising the assay buffers described above inconcentrated form. Preferably, the reagent compositions can be diluted,preferably with an aqueous solution, to result in an assay buffer havingthe optimal concentration of ingredients for use in an assay, preferablyan ECL assay.

Another embodiment relates a dry reagent precursor comprising one of theabove described assay buffers in dry form. Preferably, the dry reagentprecursor can be combined with a solution, preferably with an aqueoussolution, to result in an assay buffer solution having the optimalconcentration of ingredients for use in an assay, preferably an ECLassay.

Another aspect of the invention relates to a reagent containing one ormore pH buffers substantially free of inorganic phosphate suitable foruse in providing a composition for conducting an assay, preferably aluminescence assay, more preferably a chemiluminescence assay or anelectrode induced luminescence assay, and most preferred anelectrochemiluminescence assay.

Another aspect of the invention relates to a reagent containing one ormore ECL assay background reducing agents (preferably, non-phosphate pHbuffering agents) suitable for use in providing a composition forconducting an assay, preferably a luminescence assay, more preferably achemiluminescence assay or an electrode induced luminescence assay, andmost preferred an electrochemiluminescence assay. Preferably, thereagent has less than 15 mM inorganic phosphate, more preferably it hasless than 5 mM inorganic phosphate, even more preferably it has lessthan 1 mM phosphate, even more preferably it is substantially free ofinorganic phosphate, and most preferably it is free of inorganicphosphate.

According to one embodiment, the reagent comprises an ECL assay bufferreducing agent (preferably, a non-phosphate pH buffering agent) and/oris substantially free of inorganic phosphate, and the reagent issuitable for use in providing a composition for conducting an ECL assaywherein electromagnetic radiation is emitted by an assay compositioncomprising members selected from the group consisting of:

(i) a metal-containing ECL moiety capable of being converted to anexcited state from which electromagnetic radiation;

(ii) an ECL co-reactant (preferably an amine or an amine moiety, mostpreferably a tertiary amine, most preferably TPA) which when oxidizedforms a strong reducing agent; and

(iii) an electrolyte capable of functioning as a medium in which saidECL moiety and said ECL co-reactant can be oxidized.

Preferably, said reagent comprises said pH buffer, said ECL co-reactantand one of the other two members of said group (i)-(iii).

Another aspect of the invention relates to assay compositions comprisingone or more binding reagents, enzymes and/or substrates and the pHbuffer of the invention.

Another aspect of the invention relates to compositions, reagents, kitsand methods for carrying out protein kinase and phosphorylase assaysand/or for measuring phospho-peptides, phospho-proteins, andphospho-amino acids. One embodiment of the invention relates to acomposition comprising a pH buffer and a phospho-peptide specificantibody, where the composition is substantially free of inorganicphosphate. Preferably, the composition is free of inorganic phosphate.Preferably, the composition further comprises a phosphopeptide,phosphoamino acid and/or phosphylated protein that binds thephospho-peptide specific antibody.

Preferably, the pH buffer is selected from the group consisting ofglycylglycine, tris[hydroxymethyl)aminomethane or combinations thereof.

Preferably, the composition further comprises one or more componentsselected from the group consisting of kinases and kinase substrate.According to another embodiment, the compositions comprise or one ormore components selected from the group consisting of phosphatase andphosphatase substrate.

Preferably, the composition has a pH between 6 to 9, preferably between7 to 8, more preferably from 7.5 to 8, and most preferred about 7.8.

According to one preferred embodiment, the composition further comprisesone or more ECL co-reactants. Preferably, the ECL co-reactant comprisesan amine or an amine moiety. More preferably, the ECL co-reactantcomprises tripropylamine (TPA).

According to another preferred embodiment, the composition furthercomprises a stop reagent. Preferably, the stop reagent comprisesethylenediaminetetraacetic acid (EDTA).

According to another preferred embodiment, the composition furthercomprises an acid or base, preferably KOH.

According to one preferred embodiment, the reagents or compositions ofthe invention further comprises one or more detergents and/orsurfactants (e.g., classes of non-ionic detergents/surfactants known bythe trade names of Brij, Triton, Tween, Thesit, Lubrol, Genapol,Pluronic, Tetronic, F108, and Span).

Preferably, the composition comprises an inhibitor and/or an enzyme,more preferably an inhibitor to and/or an enzyme for a phosphorylatingor dephosphorylating reaction.

Another embodiment of the invention relates to a composition comprisinga pH buffer and an ECL co-reactant, said composition being substantiallyfree of inorganic phosphate. Preferably, the composition is free ofinorganic phosphate.

Preferably, the pH buffer is selected from the group consisting ofglycylglycine, tris[hydroxymethyl)aminomethane or combinations thereof.

Preferably, the composition comprises one or more components selectedfrom the group consisting of kinases and kinase substrate or one or morecomponents selected from the group consisting of phosphatase andphosphatase substrate.

Preferably, the composition has a pH between 6 to 9, preferably between7 to 8, more preferably from 7.5 to 8, and most preferred about 7.8.

Preferably, the ECL co-reactant comprises an amine or an amine moiety.More preferably, the ECL co-reactant comprises tripropylamine (TPA).

According to another preferred embodiment, the composition furthercomprises a stop reagent. Preferably, the stop reagent comprisesethylenediaminetetraacetic acid (EDTA).

According to another preferred embodiment, the composition furthercomprises an acid or base, preferably KOH.

Preferably, the composition comprises an inhibitor and/or an enzyme,more preferably an inhibitor to and/or an enzyme for a phosphorylatingor dephosphorylating reaction.

According to one preferred embodiment, the pH buffer is glycylgycine andsaid composition further comprises ethylenediaminetetraacetic acid(EDTA) and TPA. Preferably, the composition also further comprises KOHand/or an ECL moiety.

Another embodiment of the invention relates to a reagent comprising a pHbuffer, wherein said pH buffer is substantially free of inorganicphosphate and said reagent is suitable for use in providing acomposition for conducting an ECL assay wherein electromagneticradiation is emitted by an assay composition comprising members selectedfrom the group consisting of:

(i) a metal-containing ECL moiety capable of being converted to anexcited state from which electromagnetic radiation is emitted;

(ii) an ECL co-reactant which when oxidized forms a strong reducingagent; and

(iii) an electrolyte capable of functioning as a medium in which saidECL moiety and said amine or amine moiety can be oxidized.

Preferably, the reagent further comprises said pH buffer, the ECLco-reactant (preferably an amine or amine moiety) and one of the othertwo members of said group (i)-(iii).

Another embodiment of the invention relates to a reagent comprising a pHbuffer, wherein said pH buffer is substantially free of inorganicphosphate and said reagent is suitable for use in providing acomposition for conducting an ECL assay wherein electromagneticradiation is emitted by an assay composition comprising members selectedfrom the group consisting of:

(i) a metal-containing ECL moiety capable of being converted to anexcited state from which electromagnetic radiation is emitted;

(ii) an ECL co-reactant which when oxidized forms a strong reducingagent, wherein said ECL co-reactants is an amine or an amine moiety(preferably TPA); and

(iii) an electrolyte capable of functioning as a medium in which saidECL moiety and said amine or amine moiety can be oxidized.

Another embodiment of the invention relates to a composition comprising:

(a) a phospho-specific antibody;

(b) a reagent selected from the group consisting of phosphorylatingenzyme, a substrate to a phosphorylating enzyme or combinations thereof,and

(b) a pH buffer,

where the composition is substantially free of, preferably free ofinorganic phosphate.

Preferably, the composition also comprises an ECL co-reactant (e.g.,TPA).

5.8 Kits

One aspect of the invention relates to kits comprising, in one or morecontainers, one or more components of the ECL assay buffers of theinvention. These components may be combined, optionally with additionalreagents, to form the ECL assay buffers of the invention. The kits mayalso comprise additional assay related components such as ECL labels,ECL labeled assay reagents, enzymes, binding reagents, electrodes, assayplates, etc.

Another aspect of the invention relates to kits containing, in one ormore containers, one or more ECL assay buffers that contain atrialkylamine coreactant of the invention other than TPA. Preferably,the kit is contained in one or more glass or plastic containers,appropriately labeled with information regarding the buffer contents andinstructions regarding proper storage and use in assay. Some or all ofthe components of the ECL assay buffer may be stored in a dry state. Thekits may further comprise other assay related components such asenzymes, binding reagents, electrodes, assay plates, etc.

Another aspect of the invention relates to kits containing, in one ormore containers, one or more ECL assay buffers that are substantiallyfree of inorganic phosphate and/or comprise ECL assay buffer reducingagents (preferably, non-phosphate pH buffering agents). Preferably, thekit is contained in one or more glass or plastic containers,appropriately labeled with information regarding the buffer contents andinstructions regarding proper storage and use in assay. Some or all ofthe components of the ECL assay buffer may be stored in a dry state. Thekits may further comprise other assay related components such as ECLlabels, ECL labeled assay reagents, enzymes, binding reagents,electrodes, assay plates, etc.

No formal study on shelf-life stability of Gly-Gly/TPA buffer has beenperformed. However, using 3-4 month old assay buffer did not affectassay performance. Applicants believe that the same precautions shouldbe used for Gly-Gly stability, for example, as for ORIGEN assay buffer.Preferably, concentrations of divalent ions in the solution are keptbelow the μM level.

Preferably, the kit is adapted or suitable for performing an ECL assaywherein electromagnetic radiation emitted by a composition is detected,which kit contains, in one or more containers, a pH buffer and the kitis, preferably, substantially free of inorganic phosphate and/orcomprises an ECL assay buffer reducing agent (preferably, anon-phosphate pH buffering agent). This kit also comprises at least oneother component selected from the group consisting of: (i) ametal-containing ECL moiety capable of being converted to an excitedstate from which electromagnetic radiation is emitted; (ii) an ECLco-reactant (preferably an amine or an amine moiety) which when oxidizedforms a strong reducing agent; and (iii) an electrolyte capable offunctioning as a medium in which said ECL moiety and said ECLco-reactant (e.g., amine or amine moiety) can be oxidized, said kitcomprising at least one separate component in which one or more membersof the group consisting of said ECL moiety (i), ECL co-reactant (ii),and electrolyte (iii) is included.

Another aspect of the invention relates to kits for use in conductingassays, preferably luminescence assays, more preferably electrodeinduced luminescence assays, and most preferablyelectrochemiluminescence assays, comprising, in one or more containers,one or more pH buffers substantially free of inorganic phosphate and atleast one assay component selected from the group consisting of: (a) atleast one luminescent label (preferably electrochemiluminescent label);(b) at least one ECL co-reactant; (c) one or more phospho-specificbinding reagents; (d) one or more electrodes and/or magnetic beads; (e)one or more blocking reagents; (f) preservatives; (g) stabilizingagents; (h) enzymes; (i) detergents; (j) desiccants and/or (k)hygroscopic agents.

Preferably, the kit comprises the assay module having one or more assayelectrodes, preferably an assay plate, more preferably multi-well assayplates and the assay component(s) in one or more, preferably two ormore, more preferably three or more containers according to U.S.application Ser. No. 10/185,274 (now U.S. Pat. No. 7,842,246) and Ser.No. 10/185,363 (now U.S. Pat. No. 6,977,722), each filed on Jun. 28,2002, hereby incorporated by reference.

According to one embodiment, the kit comprises one or more of the assaycomponents in one or more multi-well plate wells, preferably in dryform.

According to one embodiment, the assay components are in separatecontainers. According to another embodiment, the kit includes acontainer comprising binding reagents and stabilizing agents. Accordingto another embodiment, the well reagents may include binding reagents,stabilizing agents. Preferably, the kits do not contain any liquids inthe wells.

One preferred embodiment relates to a kit for use in conductingelectrode induced luminescence assays (preferablyelectrochemiluminescence assays) comprising an assay plate, preferably amulti-well assay plate, one or more pH buffers and at least one assaycomponent selected from the group consisting of at least one luminescentlabel (preferably electrochemiluminescent label) and at least oneelectrochemiluminescence coreactant, wherein said pH buffers comprise anECL assay buffer background reducing agent (preferably, a non-phosphatepH buffering agent) or are substantially free of phosphate and/or saidECL coreactant is not TPA (and is preferably a functionalized tertiaryalkylamine, most preferably PIPES).

Another embodiment relates to a kit comprising a multi-well plate and apH buffer and at least one electrode induced luminescent label(preferably electrochemiluminescent label) and/or at least onebioreagent and/or at least one blocking reagent (e.g., BSA), where thekit comprises an ECL assay buffer background reducing agent (preferably,a non-phosphate buffering agent), is substantially free of inorganicphosphate and/or comprises an ECL coreactant other than TPA (preferablya functionalized tertiary alkylamine, most preferably PIPES).

According to one preferred embodiment, the kit comprises at least onematerial selected from group consisting of intact cell, cell lysate,cell fragment, cell membrane, membrane ghost, organelle, organellefragment, organelle membrane, virion, virion fragment, virion membrane,liposome, detergent solubilized protein, detergent solubilized lipid orcombinations thereof.

According to another embodiment, the kit comprises a biomaterialselected from the group consisting of plasma membrane fragments,endosomes, clathrin-coated vesicles, endoplamic reticulum fragments,synaptic vesicles, golgi fragments, membrane subdomains, mitochondria,peroxisomes, lysosomes, liposomes, viral particles, viral-inducedmembrane enclosed particles shed from cells, and intact,organismally-derived lipid membrane bodies.

According to one preferred embodiment, the kit comprises at least onebioreagent, preferably immobilized on the plate surface selected from:antibodies, fragments of antibodies, proteins, enzymes, enzymesubstrates, inhibitors, cofactors, antigens, haptens, lipoproteins,liposaccharides, cells, sub-cellular components, cell receptors,viruses, nucleic acids, antigens, lipids, glycoproteins, carbohydrates,peptides, amino acids, hormones, protein-binding ligands,pharmacological agents, luminescent labels (preferably ECL labels) orcombinations thereof. Preferably, at least one bioreagent is adapted orselected to binding to one or more membranes resulting in an electrodehaving such immobilized membranes.

Preferably, the biomaterial comprises a lipid/protein layer whichcontains at least one active protein selected from the group consistingof: single transmembrane receptors with intrinsic tyrosine kinaseactivity; non-tyrosine kinase transmembrane receptors (e.g., transferrinreceptor); G-protein coupled receptors (GPCR); GPCR effector proteins(e.g., adenylate cyclase); phosphoinositides (e.g., phosphatidy inositol4,5 bisphosphate (PIP₂)); phospholipid or sphingolipid composition,identification, or function (i.e., changes in phosphotidylserinepresence during apoptosis); organelle-bound GTPases/guanine nucleotideexchange factors (CEFs)/GTPase activating proteins (CAPs);cytokine/chemokine receptors; cell adhesion molecules (e.g., VCAM,integrins); cytoplasmic peripheral membrane protein kinases (e.g., src);intracellular protein kinase adaptor/docking proteins (e.g., insulinreceptor substrate 1, CRB2); ion channels (e.g., nicotinic acetylcholinereceptor, CFTR, etc.); passive transporters (e.g., glucose); active(ATP-driven) transporters; ion-linked transporters (e.g., Na+/glucose);glycosyltranferases/glycoprotein modifying enzymes; nuclear membranefragments; and soluble receptors.

Preferably, the kit includes immobilized reagents that compriseproteins, nucleic acids, or combinations thereof.

According to one preferred embodiment, the plurality of wells includesat least two different bioreagents. For example, a well may include twoor more assay domains, wherein two or more assay domains have differentbioreagent

Preferably, the kit comprises at least one electrochemiluminescencecoreactant and/or at least one electrode induced luminescence label(preferably electrochemiluminescent label).

According to another embodiment, the kit is adapted for multiple assays.Preferably, the kit further comprises an additional assay reagent foruse in an additional assay, the additional assay selected from the groupconsisting of radioactive assays, enzyme assays, chemical colorimetricassays, fluorescence assays, chemiluminescence assays and combinationsthereof.

According to another embodiment, the kit comprises two or more,preferably four or more, more preferably eight or more, more preferably15 or more and most preferably 25 or more assay modules or plates.According to a preferred embodiment, the kit is contained in aresealable bag or container (e.g., zip-lock opening).

Preferably, the bag or container is substantially impermeable to water.According to one preferred embodiment, the bag is a foil, preferably analuminized foil.

The packaging may be translucent, transparent or opaque. Preferably, theplates are packaged in aluminum lined plastic containers or bagscontaining a dry or inert atmosphere (e.g., the bags may be sealed underan atmosphere of nitrogen or argon or the bags may contain a dessicant).According to another embodiment, the containers are vacuum sealed.

Preferably, the container contains 1 plate. According to anotherembodiment, the container contains ten plates. According to anotherembodiment, the container includes between 10 and 100 plates.

Preferably, the assay modules or plates are sterile and/or substantiallyfree of dust and other contaminants.

Preferably, the assay modules are also substantially sterile.

According to one embodiment, the kit is manufactured (at least in part)and/or packaged in a “clean room” environment. Preferably, the kit ismanufactured (at least in part) and/or packaged in a Class 100,000 cleanroom having <100,000 particles (the clean room particle count using a0.5 micron particle count number) per cubic foot (or 3.53 millionparticles per cubic meter).

Preferably, the contaminant particle counts (particles less than 0.5microns) of the kit is less than 60 million per square meter, morepreferably 30 million per square meter, even more preferably less than20 million, even more preferably less than 15 million and mostpreferably less than 10 million.

Preferably, the non-volatile residue in deionized water is less than0.50 g/meter², more preferably less than 0.25 g/meter², even morepreferably less than 0.15 g/meter² and most preferably less than 0.10g/meter².

Preferably the contaminant ion concentration is less than 50 ppm, morepreferably less than 20 ppm, even more preferably less than 10 ppm, evenmore preferably less than 5 ppm, and most preferably less than 1 ppm.

5.9 Methods

Another aspect of the present invention relates to methods of using theimproved buffers, reagents and/or compositions of the invention.

One embodiment of the invention relates to a method for conducting anelectrochemiluminescence assay wherein electrochemiluminescence isinduced in the presence of an ECL assay buffer of the invention.Preferably, the electrochemiluminescence is induced using a carbon-basedelectrode.

Another embodiment of the invention relates to a method for measuringthe quantity of an ECL label wherein the label is induced to emitelectrochemiluminescence in the presence of an ECL assay buffer of theinvention and the electrochemiluminescence is measured so as to measurethe quantity of the ECL label. Preferably the electrochemiluminescenceis induced using a carbon-based electrode. Most preferably, the label isbound to or held in proximity to the electrode.

Another embodiment of the invention relates to a method for measuringthe quantity or activity of an analyte wherein the analyte reacts with,forms a complex with, or competes in a specific binding interaction witha labeled substance that comprises an ECL label, wherein the label isinduced to emit electrochemiluminescence in the presence of an ECL assaybuffer of the invention and the electrochemiluminescence is measured soas to measure the quantity or activity of the analyte. Preferably theelectrochemiluminescence is induced using a carbon-based electrode. Mostpreferably, the presence or activity of the analyte results in the labelbeing bound to or released from an electrode (e.g., via the form actionof a specific binding complex or via a the cleavage or formation of achemical bond).

One embodiment of the invention relates to a method for conducting anelectrochemiluminescence assay wherein electrochemiluminescence isinduced in the presence of a composition comprising a pH buffer and anECL co-reactant, said composition being substantially free of inorganicphosphate and/or comprising an ECL assay buffer background reducingagent (preferably, a non-phosphate pH buffering agent).

Another embodiment of the invention relates to a method for conductingan electrochemiluminescence assay wherein electrochemiluminescence isinduced in the presence of a composition comprising a pH buffer and anECL co-reactant, wherein the ECL coreactant is a functionalizedtrialkylamine, preferably PIPES.

Another embodiment of the invention relates to a method of generatingemission of electromagnetic radiation comprising:

-   -   (a) forming a composition comprising:        -   (i) a metal-containing ECL moiety capable of being converted            to an excited state from which electromagnetic radiation is            emitted;        -   (ii) an ECL co-reactant (preferably an amine or amine            moiety) which, when oxidized, forms a strong reducing agent;        -   (iii) an electrolyte capable of functioning as a medium in            which said ECL moiety and said ECL co-reactant (e.g., amine            or amine moiety) can be oxidized; and        -   (iv) a pH buffers,        -   wherein said composition is substantially free of inorganic            phosphate, comprises an ECL background reducing agent            (preferably, a non-phosphate pH buffering agent) and/or said            ECL co-reactant is a functionalized tertiary alkylamine;    -   (b) exposing the composition under suitable conditions to an        amount of electrochemical energy effective to induce the        composition to emit electromagnetic radiation; and    -   (c) detecting emitted electromagnetic radiation.

Another embodiment of the invention relates to a method of effecting aspecific-binding assay, either qualitatively or quantitatively, in asample or composition comprising a pH buffer substantially free ofinorganic phosphate and a phospho-specific antibody. Preferably, thesample or composition further comprises an ECL co-reactant.

Another embodiment of the invention relates to a method of effecting aspecific-binding assay, either qualitatively or quantitatively, in awell having one or more assay domains with binding reagents immobilizedthereon using composition comprising a pH buffer substantially free ofinorganic phosphate. Preferably, the composition further comprises aphospho-specific antibody.

Another embodiment of the invention relates to a method of effecting aspecific-binding non-washed assay, either qualitatively orquantitatively, in a well having one or more assay domains with bindingreagents immobilized thereon using composition comprising a ECL assaybuffer that is substantially free of inorganic phosphate and/orcomprises an functionalized trialkylamine ECL coreactant.

Another embodiment of the invention relates to a method of performing anassay comprising forming a complex comprising a kinase product and aphospho-specific antibody, wherein said complex is not exposed toinorganic phosphate.

Another embodiment of the invention relates to a method of performing anassay comprising:

-   -   (a) forming a complex comprising a kinase product and a        phospho-specific antibody, wherein said complex is not exposed        to inorganic phosphate;    -   (b) inducing a metal-containing ECL moiety to emit        electromagnetic radiation; and    -   (c) detecting emitted electromagnetic radiation.

Preferably, the complex further comprises said metal-containing ECLmoiety.

Another embodiment of the invention relates to a method of generatingemission of electromagnetic radiation, which comprises the steps of:

(a) forming a composition comprising a pH buffer, said composition beingsubstantially free of inorganic phosphate, and (i) a metal-containingECL moiety capable of being converted to an excited state from whichelectromagnetic radiation is emitted; (ii) an amine or amine moietywhich, when oxidized, forms a strong reducing agent; and/or (iii) anelectrolyte capable of functioning as a medium in which said ECL moietyand said amine or amine moiety can be oxidized;

(b) exposing the composition under suitable conditions to an amount ofelectrochemical energy effective to induce the composition to emitelectromagnetic radiation; and

(c) detecting emitted electromagnetic radiation.

Another aspect of the invention relates to improved assays. Theinvention is useful, for example, in enabling the detection and/orquantitation of one or more analytes of interest. These reactionsinclude, for example, antigen-antibody interactions, ligand-receptorinteractions, DNA and RNA interactions, enzymatic reactions, and otherknown reactions. In certain embodiments, the invention relates to andmethods for qualitatively and quantitatively detecting the presence ofanalytes of interest in a multi-component sample or multi-componentsystem. (See, U.S. application Ser. No. 10/238,391, filed on Sep. 10,2002, now U.S. Pat. No. 7,858,321, which is hereby incorporated byreference.

One preferred aspect of the invention include methods involving one ormore of the following: (a) a phospho-specific antibody; (b) assayinvolving capture reagents immobilized on a solid surface comprising anelectrode or adjacent an electrode; and/or (c) assays involving lowdetection levels (and/or requiring high signal to background ratio).

The embodiments of the invention can be used to test a variety ofsamples which may contain an analyte or activity of interest. Suchsamples may be in solid, emulsion, suspension, liquid, or gas form. Theymay be, but are not limited to, samples containing or derived from, forexample, cells (live or dead) and cell-derived products, immortalizedcells, cell fragments, cell fractions, cell lysates, organelles, cellmembranes, hybridoma, cell culture supernatants (including supernatantsfrom antibody producing organisms such as hybridomas), waste or drinkingwater, food, beverages, pharmaceutical compositions, blood, serum,plasma, hair, sweat, urine, feces, tissue, biopsies, effluent, separatedand/or fractionated samples, separated and/or fractionated liquids,organs, saliva, animal parts, animal byproducts, plants, plant parts,plant byproducts, soil, minerals, mineral deposits, water, water supply,water sources, filtered residue from fluids (gas and liquid), swipes,absorbent materials, gels, cytoskeleton, protein complexes,unfractionated samples, unfractionated cell lysates, endocrine factors,paracrine factors, autocrine factors, cytokines, hormones, cellsignaling factors and or components, second messenger signaling factorsand/or components, cell nucleus/nuclei, nuclear fractions, chemicals,chemical solutions, structural biological components, skeletal(ligaments, tendons) components, separated and/or fractionated skeletalcomponents, hair, fur, feathers, hair fractions and/or separations,skin, skin samples, skin fractions, dermis, endodermis, eukaryoticcells, prokaryotic cells, fungus, yeast, antibodies, antibody fragments,immunological factors, immunological cells, drugs, therapeutic drugs,oils, extracts, mucous, fur, oils, sewage, environmental samples,organic solvents or air. The sample may further comprise, for example,water, organic solvents (e.g., acetonitrile, dimethyl sulfoxide,dimethyl formamide, n-methyl-pyrrolidone or alcohols) or mixturesthereof.

Analytes that may be measured include, but are not limited to, wholecells, cell surface antigens, subcellular particles (e.g., organelles ormembrane fragments), viruses, prions, dust mites or fragments thereof,viroids, antibodies, antigens, haptens, fatty acids, nucleic acids (andsynthetic analogs), proteins (and synthetic analogs), lipoproteins,polysaccharides, inhibitors, cofactors, haptens, cell receptors,receptor ligands, lipopolysaccharides, glycoproteins, peptides,polypeptides, enzymes, enzyme substrates, enzyme products, secondmessengers, cellular metabolites, hormones, pharmacological agents,synthetic organic molecules, organometallic molecules, tranquilizers,barbiturates, alkaloids, steroids, vitamins, amino acids, sugars,lectins, recombinant or derived proteins, biotin, avidin, streptavidin,or inorganic molecules present in the sample. Activities that may bemeasured include, but are not limited to, the activities ofphosphorylases, phosphatases, esterases, trans-glutaminases, nucleicacid damaging activities, transferases, oxidases, reductases,dehydrogenases, glycosidases, ribosomes, protein processing enzymes(e.g., proteases, kinases, protein phophatases, ubiquitin-proteinligases, etc.), nucleic acid processing enzymes (e.g., polymerases,nucleases, integrases, ligases, helicases, telomerases, etc.), cellularreceptor activation, second messenger system activation, etc.

Whole cells may be animal, plant, or bacteria, and may be viable ordead. Examples include plant pathogens such as fungi and nematodes. Theterm “subcellular particles” is meant to encompass, for example,subcellular organelles, membrane particles as from disrupted cells,fragments of cell walls, ribosomes, multi-enzyme complexes, and otherparticles which can be derived from living organisms. Nucleic acidsinclude, for example, chromosomal DNA, plasmid NA, viral DNA, andrecombinant DNA derived from multiple sources. Nucleic acids alsoinclude RNA's, for example messenger RNA's, ribosomal RNA's and transferRNA's. Polypeptides include, for example, enzymes, transport proteins,receptor proteins, and structural proteins such as viral coat proteins.Preferred polypeptides are enzymes and antibodies. Particularlypreferred polypeptides are monoclonal antibodies. Hormones include, forexample, insulin and T4 thyroid hormone. Pharmacological agents include,for example, cardiac glycosides. It is of course within the scope ofthis invention to include synthetic substances which chemically resemblebiological materials, such as synthetic polypeptides, synthetic nucleicacids, and synthetic membranes, vesicles and liposomes. The foregoing isnot intended to be a comprehensive list of the biological substancessuitable for use in this invention, but is meant only to illustrate thewide scope of the invention.

The composition or reagent of the invention are preferably aqueous. Thecomposition or reagent can also be non-aqueous. Examples of suitableorganic liquids are acetonitrile, dimethylsulfoxide (DMSO),dimethylformamide (DMF), methanol, ethanol, and mixtures of two or moreof the foregoing. Illustratively, tetraalkylammonium salts, such astetrabutylammonium tetrafluoroborate, are soluble in organic liquids andcan be used with them to form non-aqueous electrolytes.

Also, typically, the analyte of interest is present at a concentrationof 10⁻³ molar or less, for example, at least as low as 10⁻¹⁸ molar. Thesample which may contain the analyte of interest, can be in solid,emulsion, suspension, liquid, or gas form, and can be derived from, forexample, cells and cell-derived products, water, food, blood, serum,hair, sweat, urine, feces, tissue, saliva, oils, organic solvents orair. The sample can further comprise, for example, water, acetonitrile,dimethyl sulfoxide, dimethyl formamide, n-methyl-pyrrolidone oralcohols.

In one embodiment, the reagent includes an ECL moiety conjugated to anantibody, antigen, nucleic acid, hapten, small nucleotide sequence,oligomer, ligand, enzyme, or biotin, avidin, streptavidin, Protein A,Protein a, or complexes thereof or other secondary binding partnercapable of binding to a primary binding partner through proteininteractions.

One embodiment of the invention relates to a method of detecting orquantitating an analyte of interest by ECL assay, which comprises:

(1) forming a composition comprising

-   -   (a) a sample to be tested for the analyte of interest,    -   (b) at least one substance selected from the group consisting of        -   (i) additional analyte of interest or an analog of the            analyte of interest,        -   (ii) a binding partner of the analyte of interest or its            said analog, and        -   (iii) a reactive component capable of binding with (i) or            (ii),    -   (c) a metal-containing ECL moiety capable of being converted to        an excited state from which electromagnetic radiation is        emitted, said ECL moiety being capable of entering into a        binding interaction with the analyte of interest or a substance        defined in (b)(i), (b)(ii), or (b)(iii);    -   (d) an ECL co-reactants (preferably an amine or an amine moiety)        which, when oxidized, forms a strong reducing agent, and    -   (e) an electrolyte capable of functioning as a medium in which        said ECL moiety and said species can be oxidized;

(2) exposing said composition to an amount of electrochemical energyeffective to induce the composition to emit electromagnetic radiation;and

(3) detecting emitted electromagnetic radiation,

wherein the sample is not exposed to inorganic phosphate detrimental tothe performance of the assay or wherein said composition furthercomprises an ECL assay buffer background reducing agent (preferably, anon-phosphate pH buffering agent). Preferably, the composition has lessthan 15 mM inorganic phosphate, more preferably it has less than 5 mMinorganic phosphate, even more preferably it has less than 1 mMphosphate, even more preferably it is substantially free of inorganicphosphate, most preferably it is free of inorganic phosphate.

Solid phase assay formats (e.g., solid phase binding assays) oftencouple a biological activity or binding reaction to attachment ordissociation of a label from a surface. For example the bindinginteraction between a binding reagent that is attached and a labeledanalyte results in the localization of the label on the solid phasesupporting the immobilized binding reagent. The biological activity orbinding reaction to be measured can be quantified through a measurementof the labels on the solid phase. Many solid phase assay formats involvea wash step to remove unbound labels prior to detecting labels on thesolid phase (washed assays). Assays without this wash step can beachieved when the detection method can discriminate between free andbound labels. Non-wash assay formats are desired because washing steps,in many applications, can be difficult or cumbersome to carry out. Inmany cases, however, the performance of non-wash assays is limited byhigh background signals due to incomplete discrimination of free vs.bound labels.

We have found, surprisingly, that the ECL assay buffers of the inventionimprove the discrimination of free vs. bound labels in ECL assays usingassay reagents attached (e.g., by covalent interactions, specificbinding interaction, non-specific adsorption, etc,) to the workingelectrode used to induce ECL (i.e., the ability to selectively detectlabels that are bound to the electrode). More specifically, thecompositions and reagents of the invention improve the ratio of ECLsignal from bound label to ECL signal from free label. It is believedthat the ECL assay buffers of the invention decrease the distance fromthe solid electrode surface from which an ECL label is induced to emitluminescence. This, in turn, increases the signal of bound label (whichmay be bound to the electrode surface) vs. free label (which is notbound to the electrode). Another way to characterize this is in terms ofan “effective excitation length”—the maximum distance at which a freeECL label is able to be excited. The “effective excitation length” isimpacted by i) the distance short-lived intermediates involved in thegeneration of ECL (e.g., oxidation product of TPA) can diffuse from theelectrode before they are destroyed in a destructive side reaction (afunction of the lifetimes and diffusion constants for theseintermediates) and ii) the rate at which free labels or labeled reagentsdiffuse into the region close enough to the electrode to participate ina reaction with these reactive intermediates (a function of thediffusion constant for the unbound ECL labels or labeled reagents).

Using the ECL assay buffers of the invention, the effective excitationlength is reduced by >50%, preferably by >75%, even more preferablyby >90%. Thus, the ECL assay buffers of the invention are desirablesince they maximize the ratio of bound/free ECL signal which enhancesthe performance of the assay. These considerations are particularlyimportant for measuring low affinity interactions, which require thepresence of the labeled species in high concentration in the solutionbut would also be expected to suffer from significant signal loss due tobinding complex dissociation during wash steps.

Accordingly, another aspect of the invention relates to non-wash formatassays using pH buffer substantially free of inorganic phosphate whichmaximizes the ratio of bound/free ECL signal. Preferably, the assayinvolves the capture of an ECL label at a surface having or beingadjacent to an electrode surface. See, for example, U.S. Pat. Nos.6,066,448; 6,090,545; 6,140,045; 6,207,369, 6,214,369; and U.S.application Ser. No. 10/185,274 (now U.S. Pat. No. 7,842,246) and Ser.No. 10/185,363 (now U.S. Pat. No. 6,977,722), each filed on Jun. 28,2002, hereby incorporated by reference.

Thus, another embodiment of the invention relates generally toelectrochemiluminescence assays using reagents immobilized on a surface(preferably an electrode surface) and having advantageous effectiveexcitation lengths. Preferably, the assay results in an effectiveexcitation length less then 100 microns, more preferably less than 75microns, even more preferably less than 50 microns, even more preferablyless than 25 microns, even more preferably less than 10 microns, evenmore preferably less than 5 microns and most preferably less than 1micron. According to a particularly preferred, embodiment, the effectiveexcitation length is less than 0.5 micron, preferably less than 0.2microns, even more preferably less than 0.1 micron.

5.10 Systems

Yet another aspect of the present invention relates to system forperforming assays and comprising or using the reagents and/orcompositions of the invention.

One embodiment of the invention relates to a system for ECL detection orquantitation of an analyte of interest in a sample, said systemcomprising:

(a) a pH buffering agent;

(b) a sample;

(c) at least one substance selected from the group consisting of:

-   -   -   (i) added analyte of interest or an analog of the analyte of            interest,        -   (ii) a binding partner of the analyte of interest or its            said analog, and        -   (iii) a reactive component capable of binding with (i) or            (ii),

wherein one of said substances is linked, either directly or through oneor more other molecules, to a metal-containing ECL moiety which iscapable of being converted to an excited state from whichelectromagnetic radiation is emitted;

(d) an ECL co-reactant, preferably an amine or amine moiety, which iscapable of being converted to a strong reducing agent and anelectrolyte;

(d) one or more electrodes for inducing the ECL moiety to emitelectromagnetic radiation; and

(e) one or more detectors for measuring the emitted radiation todetermine the presence or quantity of the analyte of interest in thesample;

Wherein said pH buffering agent is substantially free of phosphate or isan ECL assay buffer background reducing agent and/or said ECL coreactantis a functionalized tertiary amine

Another embodiment of the invention relates to a system for ECLdetection or quantitation of an analyte of interest in a sample, saidsystem comprising:

(a) a pH buffering agent;

(b) a sample;

(c) at least one substance selected from the group consisting of:

-   -   -   (i) added analyte of interest or an analog of the analyte of            interest,        -   (ii) a binding partner of the analyte of interest or its            said analog, and        -   (iii) a reactive component capable of binding with (i) or            (ii),

wherein one of said substances is linked, either directly or through oneor more other molecules, to a metal-containing ECL moiety which iscapable of being converted to an excited state from whichelectromagnetic radiation is emitted;

(d) an ECL co-reactant, preferably a functionalized tertiary amine,which is capable of being converted to a strong reducing agent and anelectrolyte;

(d) one or more electrodes for inducing the ECL moiety to emitelectromagnetic radiation; and

(e) one or more detectors for measuring the emitted radiation todetermine the presence or quantity of the analyte of interest in thesample.

5.11 Method of Selecting Biologically Active Compounds and ProducingNovel Drug

Another aspect of the invention relates to improved methods and systemsfor selecting or identifying biologically active compounds and,optionally, incorporating such biologically active compounds intosuitable carrier compositions in appropriate dosages as described inparagraph 6.11 of U.S. application Ser. No. 10/185,274 (now U.S. Pat.No. 7,842,246) and Ser. No. 10/185,363 (now U.S. Pat. No. 6,977,722),each filed on Jun. 28, 2002, hereby incorporated by reference.

One embodiment relates to the use of the invention to screen for newdrugs, preferably, by high-throughput screening (HTS), preferablyinvolving screening of greater than 50, more preferably 100, morepreferably 500, even more preferably 1,000, and most preferably 5,000.According to a particularly preferred embodiment, the screening involvesgreater than 10,000, greater than 50,000, greater than 100,000, greaterthan 500,000 and/or greater than 1,000,000 compounds.

Advantageously, the reagents, compositions, methods, apparatus and/orassay plates or modules of the invention may be integrated into and/orused in a variety of screening and/or drug discovery methods. Suchscreening and/or drug discovery methods include those set forth in U.S.Pat. No. 5,565,325 to Blake; U.S. Pat. No. 5,593,135 to Chen et al.;U.S. Pat. No. 5,521,135 to Thastrup et al.; U.S. Pat. No. 5,684,711 toAgrafiotis et al.; U.S. Pat. No. 5,639,603 to Dower et al.; U.S. Pat.No. 5,569,588 to Ashby et al.; U.S. Pat. No. 5,541,061; U.S. Pat. No.5,574,656; and U.S. Pat. No. 5,783,431 to Peterson et al.

According to another embodiment, the invention further comprisesidentifying adverse effects associated with the drug and storinginformation relating to the adverse effects in a database. See, U.S.Pat. No. 6,219,674 by Classen.

Another aspect of the invention relates to improved biologically activecompounds and/or drugs made using the inventive methods.

6. EXAMPLES

The following examples are illustrative of some of the electrodes,plates, kits and methods falling within the scope of the presentinvention. They are, of course, not to be considered in any waylimitative of the invention. Numerous changes and modification can bemade with respect to the invention by one of ordinary skill in the artwithout undue experimentation.

Example I ECL Measurements

Unless otherwise indicated, ECL measurements were carried out usingmulti-well plates having integrated carbon ink electrodes (see, Example6.1 and, in particular, Plate Types A and B of U.S. application Ser. No.10/185,274 (now U.S. Pat. No. 7,842,246) and Ser. No. 10/185,363 (nowU.S. Pat. No. 6,977,722), each filed on Jun. 28, 2002, herebyincorporated by reference). The electrodes were, optionally treated withan oxygen plasma prior to being coated with binding reagents (plasmatreated and non-plasma treated plates, respectively, are designatedhereafter as PT or NPT plates). Binding reagents were immobilized on theworking electrodes of the plates using the methods described in the U.S.application Ser. No. 10/185,274 (now U.S. Pat. No. 7,842,246) and Ser.No. 10/185,363 (now U.S. Pat. No. 6,977,722), or adaptations thereof.Unless otherwise indicated, ECL measurements were carried out usingmulti-well plate readers adapted for use with these multi-well plates.The readers and their use are described in Example 6.3 of the U.S.application Ser. No. 10/185,274 (now U.S. Pat. No. 7,842,246) and Ser.No. 10/185,363 (now U.S. Pat. No. 6,977,722). These applications and, inparticular, the descriptions of plate types, immobilization methods,plate readers and ECL measurement methods disclosed therein, are herebyincorporated by reference. The reported ECL intensities are reported inrelative terms and may depend on the instrument, gain settings andplates used in a particular experiment. For this reason, the absolutevalues reported in different experiments may not be directly comparable.

Example II Tyrosine Kinase Assay

The format involved the phosphorylation of a kinase substrateimmobilized on electrodes in multi-well plates adapted for ECLmeasurements (see Example 1), complexation of the product to a labeledanti-phosphotyrosine antibody and detection of the surface-bound labelvia an ECL measurement in the presence of an ECL Assay Buffer comprisingan ECL coreactant. The electrodes were pretreated by etching in anoxygen plasma to increase the amount of exposed carbon. The kinasesubstrate—poly(Glu, Tyr) having a 4:1 ratio of Glu to Tyr and amolecular weight of 20,000-50,000 Daltons (PGT, Sigma-Aldrich Co.)—wasimmobilized by non-specific adsorption on the surface of the workingelectrodes in the wells of the plates. The working electrode in eachwell was treated with 1500 nL of a solution containing 1 mg/ml PGT inPBS buffer. The plate was then dried overnight and blocked in a 5%solution of bovine serum albumin at 4° C. The plate was washed to removethe blocking agent prior to use.

The assay was carried out by adding, to each well, 50 μL of a bufferedsolution containing a soluble tyrosine kinase (c-src, UpstateBiotechnology), an anti-phosphotyrosine monoclonal antibody (Abzyme,IGEN International) that was labeled with a sulfonated derivative ofruthenium-tris-bipyridine (Sulfo-TAG™ label, Meso Scale Discovery), ATPand Mg⁺². The reaction was allowed to proceed for 1 hour. The plate waswashed and 100 μL of an ECL Assay Buffer containing tripropylamine wasadded. The plate was analyzed using electrochemiluminescence detectionas described in Example I.

When a conventional ECL Assay Buffer containing TPA in a phosphatebuffer (ORIGEN Assay Buffer, IGEN International) was used in theprotocol, the complex formed between the labeled antibody and thephosphorylated substrate dissociated over a period of 30 min. to an hour(the majority of the dissociation occurring within the first fewminutes) due to the competitive binding of phosphate ions with thelabeled antibodies. One approach to avoiding this problem is to controlthe time of exposure of the formed complex to the inorganicphosphate-containing solution. This approach, however, may beimpractical in some assays such as high throughput assays involvinglarge numbers of plates.

Applicants discovered another solution to overcome the problem was theuse of phosphate-free buffers. Surprisingly, it was discovered that thephosphate could be replaced with other buffers without compromising theability of the ECL Assay Buffers to support the generation of ECL.

Assays were carried out using the following two ECL Assay Buffercompositions:

Gly-Gly Assay Buffer:

0.4 M Glycylglycine (Gly-Gly)

0.1 M Tripropylamine (TPA)

12 mM Ethylenediaminetetraacetic Acid (EDTA)

pH=7.8 (adjusted by addition of 10% KOH)

Tris Assay Buffer:

0.4 M Tris(hydroxymethyl)aminomethane (Tris)

0.15 M Tripropylamine (TPA)

12 mM Ethylenediaminetetraacetic Acid (EDTA)

pH 7.8 (adjusted by addition of 10% KOH)

EDTA was added into the new ECL Assay Buffers to stop thephosphorylation reaction by sequestering Mg⁺², an ion required forkinase activity (EDTA was not required in phosphate-based ECL AssayBuffers due to the affinity of phosphate for magnesium ions). Thiscomposition allowed us to combine two steps (addition of stop-solutionand actual assay buffer) into one step. Applicants found that EDTAinterfered with ECL generation at concentrations higher that 10 mM, butthat 5-12 mM EDTA was enough to stop the reaction while only causing asmall decrease in ECL signal.

FIG. 1 shows the decrease in ECL from the phosphopeptide-antibodycomplex as a function of the time between the addition of the AssayBuffer and the measurement of ECL. Surprisingly, while exposure of thecomplex to the phosphate-containing ORIGEN Assay Buffer led to almostcomplete dissociation of the complex (within the time it took to put theplate into the ECL reader for the 0 min. point), the complex showedexcellent stability in the Gly-Gly (<80% dissociation over 40 min) andTris (<55% dissociation over 40 min) Assay Buffers.

The stability of the complex was improved further by eliminating thewash step prior to addition of the assay buffer. FIG. 2 shows theresults of an experiment in which 200 μL of Gly-Gly Assay Buffer (asdescribed above except that the concentration of EDTA was 5 mM and 0.2%Triton X-100 was added) was added directly to the reaction mixturewithout an intervening wash step. Storage of the plates for 20 hoursprior to measuring ECL resulted in only a 15% decrease in signal.

One additional surprising advantage of the protocol was its robustness.Surprisingly, the time of the phosphorylation step was the only timethat required tight control in order to get reproducible results. Theresults of the assay did not depend on the time between all other steps.

Example III Detection of Phosphorylated EGF Receptor

A sandwich immunoassay was used to detect autophosphorylated α-epidermalgrowth factor receptor (α-EGFR) in cell lysates prepared fromEGF-activated A-431 cells (American Type Culture Collection). The assayemployed a biotin labeled capture antibody directed against the α-EGFRextracellular domain and a Sulfo-TAG labeled detection antibody that isspecific for phosphotyrosine (see Example II). The biotin-labeledantibody was immobilized on the working electrode of multi-well platesadapted for use in ECL assays (see Example I) through the interaction ofthe biotin label with avidin that was passively adsorbed on theelectrode surface. Solubilized EGFR (in RIPA, a deoxycholate-containingbuffer) was then added and allowed to bind to the anti-EGFR antibody.Subsequently, the Sulfo-TAG labeled α-phosphotyrosine antibody was addedto detect autophosphorylated EGFR.

In an end-product stability experiment, an assay was carried out asdescribed above and, prior to the induction and measurement of ECL, theresulting sandwich complex was incubated for varying amounts of time intwo different ECL Assay Buffers: 150 mM TPA/150 mM Phosphate, pH 7.48and 100 mM TPA/400 mM glycine-glycine pH 7.8. FIG. 3 shows that therewas a significant time-dependent decay in signal in the presence of thephosphate-containing buffer; the signal decreased by roughly 80% afterone hour. The glycine-glycine buffer reduced this decrease to roughly20%. The great loss of signal that occurs in the phosphate buffer isbelieved to be due to the phosphate ion competing with thephosphorylated protein for the anti-phosphotyrosine antibody. Moreover,the signal to background ratio was increased 2.5 fold using theglycyl-glycine assay buffer.

Example IV Effect of ECL Assay Buffer Composition on the Ability toDiscriminate Between Specific Signal and Assay Buffer Background

In many ECL assay formats, the sensitivity with which an ECL label canbe measured is limited by the light signal (and the noise in the lightsignal) generated by the ECL Assay Buffer in the absence of the ECLlabel (ECL Assay Buffer background). This limitation is especiallyevident in washed assays, assays exhibiting low levels of non-specificbinding and/or assays employing ECL readers having sensitive, low noise,light detectors. Applicants examined the relationship between ECL AssayBuffer composition and the ability to discriminate between the signaldue to an ECL label and the ECL Assay Buffer background. In particular,applicants tested four ECL Assay Buffer formulations that varied in theidentity of the ECL coreactant and/or the identity of the pH bufferingagent: TPA/Phosphate, TPA/Tris, TPA/Gly-Gly and PIPES/Phosphate (wherePIPES stands for 1,4-piperazine-1,4-bis(2-ethanesulfonic acid).

The experiments were carried out on multi-well plates (as described inExample I) that had avidin immobilized on the working electrodes. Theexperiments were carried out on plates that were treated with an oxygenplasma (PT plates) as well as on untreated plates (NPT plates). Avidinwas immobilized on PT plates by dispensing 2.5 uL of solution containing0.5 mg/mL avidin and 0.0035% Triton X-100 on the working electrode ofeach well, allowing the solution to evaporate to dryness over a periodof 1 hour and blocking the remaining surfaces of the well overnight at4° C. with a 5% (w/v) solution of BSA. Avidin was immobilized on NPTplates by dispensing 2.5 uL of solution containing 0.5 mg/mL avidin and0.0075% Triton X-100 on the working electrode of each well, allowing thesolution to evaporate to dryness overnight and blocking the remainingsurfaces of the well for 2 hours with a 5% (w/v) solution of BSA.Varying amounts of an ECL label could be brought into proximity with theelectrode surface by treating the wells with a solution containingbovine IgG that was labeled with biotin and ˜1.9 Sulfo-TAG labels perprotein (BT-IgG*). The binding of the BT-IgG* was accomplished by adding50 uL of a solution containing 1 nM of BT-IgG* in PBS to the wells andincubating for a period of 60 minutes while shaking. The wells werewashed with water, 100 uL of ECL Assay Buffer was added and ECL wasmeasured. The signal due to ECL Assay Buffer Background was measured byrepeating the experiment as described above except that the BT-IgG* wasomitted.

The four combinations of coreactant and pH buffer to be tested wereoptimized to identify the concentration of coreactant, the concentrationof pH buffer and the pH that gave the best ratio of signal from BT-IgG*to ECL Assay Buffer background (S/B ratio). The concentrations ofcoreactant were varied from 25 to 200 mM for TPA or 13 to 200 mM forPIPES. The concentrations of pH buffer were varied from 50 to 300 mM forphosphate, 100-600 mM for Tris or 50-800 mM for Gly-Gly. The pH wasvaried from 7 to 8. In all cases, the ECL Assay Buffers also contained0.05% Triton X-100. KOH or HCl were added as necessary to achieve thedesired pH. Within the ranges tested, all the formulations gave adequateperformance for use in ECL assays, however, the following optimizedformulations were identified on the basis of their S/B ratios:TPA/Phosphate (125 mM TPA, 200 mM phosphate, 0.05% Triton X-100, pH7.5); TPA/Tris (125 mM TPA, 200 mM Tris, 0.05% Triton X-100, pH 7.8);TPA/Gly-Gly (100 mM TPA, 200 mM Gly-Gly, 0.05% Triton X-100, pH 7.8) andPIPES/Phos (25 mM PIPES, 100 mM phosphate, 0.05% Triton X-100, pH 7.5).

FIGS. 4A and 4B compare the performance of the four optimizedformulations for assays carried out on NPT plates and PT plates,respectively. We found that the TPA/Phosphate and TPA/Tris buffers gaveroughly comparable signals for the BT-IgG*, however, the lower ECL AssayBuffer Background of the TPA/Tris system led to a significantimprovement in the S/B ratio relative to the TPA/Phosphate buffer.Assuming the noise in the background to be roughly proportional to thebackground signal, the 4-6 fold improvement in S/B ratio transfersdirectly to a 4-6 fold improvement in detection limits. Despite havinglower specific signals, the TPA/Gly-Gly buffer had an S/B ratio that wasapproximately the same as the TPA/Tris buffer and could, therefore, beexpected to produce similar detection limits. The PIPES/Phosphate bufferperformed slightly better (in terms of S/B ratio) than the TPA/Phosphatebuffer on unetched plates and slightly worse on etched plates.

Example V Effect of the ECL Assay Buffer Composition on the Ability toDiscriminate Between ECL Labels that are Bound to an Electrode Surfaceand ECL Labels that are Free in Solution

In some ECL assay formats, the sensitivity with which an ECL label heldin proximity to an electrode can be measured is limited by the lightsignal (and the noise in the light signal) generated by ECL labels insolution. This limitation is especially evident in assays in whichlabels in solution are not removed by washing prior to the addition ofan ECL Assay Buffer and the measurement of ECL (Unwashed Assays).Applicants examined the relationship between ECL Assay Buffercomposition and the ability to discriminate between the signal due toECL labels bound to (or held in proximity to) an electrode and ECLlabels that are free in solution.

In these experiments, the specific signal from bound ECL labels wasmeasured using BT-IgG* bound to avidin-coated electrodes as described inExample IV. The ECL Assay Buffer background was determined by omittingthe BT-IgG*, also as described in Example IV. The ECL signal from freeECL labels in solution was determined similarly to the ECL Assay Bufferbackground except that the ECL Assay Buffer added to the wells included10 nM bovine IgG having 3.9 labels per protein (IgG*). The ratio of theECL signal from the bound BT-IgG* to the ECL signal from the free IgG*(B/F ratio) is indicative of the sensitivity with which bound ECL labelscan be detected in the presence of free ECL labels in solution.

The four optimized ECL Assay Buffers from Example IV were tested fortheir ability to discriminate between surface bound ECL labels. Theresults are presented in Tables IA and IB. The replacement of phosphatewith Tris led to some improvement in the B/F ratio for TPA-containingbuffers. The most drastic improvement, however, was achieved bysubstituting the coreactant component, i.e., by replacing TPA withPIPES. There was a 4-5 fold improvement in the B/F ratio by replacingTPA/Phos with PIPES/Phos.

TABLE IA ECL signal measured on avidin-coated PT plates from boundBT-IgG* (bound from a 1.5 nM solution), free IgG* (present in a 1.5 nMsolution) and ECL Assay Buffer Background. Bound BT-IgG* Free IgG*Background S/B B/F TPA/Phosphate 77493 2267 305 254 39 TPA/Tris 831671873 61 1363  46 TPA/Gly-Gly 28168 1111 35 805 26 PIPES/Phosphate 64724670 319 203 183 S/B = (Bound)/(Background); B/F = (Bound −Background)/(Free − Background).

TABLE IB ECL signal measured on avidin-coated NPT plates from boundBT-IgG* (bound from a 1.5 nM solution), free IgG* (present in a 1.5 nMsolution) and ECL Assay Buffer Background. Bound BT-IgG* Free IgG*Background S/B B/F TPA/Phosphate 264,063 4671 464  569 63 TPA/Tris270,809 2734 89 3043 102 TPA/Gly-Gly 123,393 1663 38 3247 76PIPES/Phosphate 172,226 728 164 1050 305 S/B = (Bound)/(Background); B/F= (Bound − Background)/(Free − Background).

PIPES-containing ECL Assay Buffers were prepared with phosphate, Tris orGly-Gly as the buffering agent. The B/F ratio of each of these mixtureswas further optimized by varying the concentration of PIPES and thebuffer component. The concentration of PIPES was varied from 12.5 to 200mM in the phosphate-based buffer and 25 to 100 mM in the Tris andGly-Gly buffers. The concentrations of the buffering agent were variedfrom 100 to 400 mM. In all cases, the ECL Assay Buffers also contained0.05% Triton X-100. KOH or HCl were added as necessary to achieve thedesired pH. Within the ranges tested, all the formulations gave adequateperformance for use in ECL assays including compositions that had noadded buffer component. It was also possible to omit the buffering agentand achieve adequate performance due the ability of PIPES to act as a pHbuffer. PIPES concentrations of 20-100 mM were found to provide high B/Fratios while maintaining reasonable ECL intensities. The bestperformance was achieved when the phosphate concentrations was roughly2-4 times the PIPES concentration. The following optimized formulationswere identified on the basis of having both high S/B ratios andreasonable signal intensities: PIPES/PHOSPHATE (25 mM PIPES, 100 mMphosphate, 0.05% Triton X-100, pH 7.5); PIPES/Tris (25 mM PIPES, 200 mMTris, 0.05% Triton X-100, pH 7.8) and PIPES/Gly-Gly (25 mM PIPES, 100 mMGly-Gly, 0.05% Triton X-100, pH 7.8).

FIGS. 5A and 5B compare the performance of the three optimizedformulations for nonwashed assays carried out on NPT plates and PTplates, respectively. The figures compare the performance to theconventional TPA/Phosphate buffer. We found that for all three bufferingagents that were tested, the use of PIPES as a coreactant led tosignificant improvements (as much as factors of 4-5) in the B/F ratiorelative to TPA/Phosphate.

Example VI Effect of Detergent on the Performance of ECL Assay Buffers

FIG. 6 shows the effect of the presence of various non-ionic detergentson ECL signal from BT-IgG* bound to avidin-coated plasma treatedelectrodes. The detergents were added at 0.5% (w/v) to one of two ECLAssay Buffers: FIG. 6A shows the results obtained with 150 mM TPA, 250mM phosphate, pH 7.5; FIG. 6B shows the results obtained with 50 mMPIPES, 150 mM phosphate, pH 7.5. BT-IgG* (50 μL of a 0.01 nM solution)was allowed to bind to the avidin surface. The plates were washed, ECLAssay Buffers were added and the plates were analyzed using ECLdetection. The figure shows the Assay Buffer background, signal and S/Bratio (calculated as in Example 4) measured using each of the ECL AssayBuffers. Of the detergents tested, only Triton X-100 had a significanteffect on performance. For the PIPES-based buffer, the effect was large;addition of Triton led to a >2.5 fold increase in the S/B ratio. Theeffect of Triton X-100 on the TPA-based buffer was much smaller. TritonX-100 differs from the other detergents present in that it contains aphenol ether moiety. Applicants hypothesize that the beneficial effectof Triton X-100 may result from the oxidation of the phenol ether moietyat the electrode and the participation of the Triton oxidation productin the ECL reaction.

Surprisingly, the effect of detergents on assays conducted on non-plasmatreated plates was different and much greater in magnitude. FIG. 7 showsthe effect of five different non-ionic detergents on TPA/Phos, TPA/Tris,TPA/Gly-Gly and PIPES/Phos Assay Buffers (the optimized formulations ofExample IV except for the composition and amount of detergent). Tween20, Thesit, Triton X-100 and Triton X-114 were all present at aconcentration of 0.05% (v/v). β-Octyl glucopyranoside was present at aconcentration of 4% (v/v). In this experiment, streptavidin-coatedelectrodes were treated with 0.018 pmol of BT-IgG* (6.3 labels perprotein) in a volume of 50 uL. The figure shows that all the detergentssignificantly improved the ECL signal measured in the presence ofTPA-containing Assay Buffers relative to the same Assay Buffer in theabsence of detergent. In most cases, the improvement was greater than 2fold. In additional experiments, it was observed that the maximalsignals observed in each Assay Buffer tended to occur at the criticalmicellar concentration (cmc) of a detergent or higher. In contrast tothe TPA-containing ECL Assay Buffers, the performance of PIPES wasimproved ˜30 fold by the addition of the phenol ether containingdetergents (Triton X-100 and Triton X-114) but very little improvementin signal was observed in the presence of the Tween and Thesitdetergents.

Applicants hypothesize that the effect of the Triton detergents on thePIPES/Phos buffer may be related to the participation of Tritonoxidation products in the ECL process. By contrast, the effect ofdetergents on the ECL signal from TPA-containing Assay Buffers on NPTelectrodes appears to be a more general phenomenon and may relate to thestabilization of TPA oxidation products in detergent micelles.

A larger screen of detergents was conducted to identify other detergentsthat improved the ECL signal from BT-IgG* on streptavidin-coated NPTelectrodes in the presence of TPA/Phosphate. The addition of all thenon-ionic detergents (APO-14, Triton X-100, β-nonyl-glucoside, Tween 20,Genapol and pentaethylene glycol mono-n-dodecyl ether) and zwitterionicdetergents (ASB-14 and Empigen) that were tested produced increases inthe ECL signal.

Example VII ECL Activity of Selected Tertiary Amines

A number of tertiary amines were screened for their ability act ascoreactants. The measurements were conducted in a similar fashion as themethods described in Examples IV and V. ECL Assay Buffers were preparedthat contained the selected tertiary amine (200 mM), phosphate buffer(200 mM), Triton X-100 (0.1%) and that were adjusted to pH 7.5. Thesignal from label attached to an electrode was measured using thefollowing procedure: i) a solution containing 0.3 nM Bt-IgG* (IgGlabeled with Sulfo-TAG and biotin) in an ECL Assay Buffer was introducedinto the wells of streptavidin-coated 96-well NPT or PT plates; ii) theplates were incubated for 2 h with shaking to allow the Bt-IgG* to bindthe surface; iii) the plates were washed four times and 150 uL of theECL Assay Buffer was added and iv) the ECL from the label was measured.The assay buffer background was measured by introducing 150 uL of an ECLAssay Buffer into a streptavidin-coated plate and measuring the ECL. TheECL signal from free ECL labels in solution was measured by introducinga solution containing 10 nM IgG* (IgG labeled with Sulfo-TAC but notbiotin) into the wells of streptavidin-coated 96-well NPT or PT platesand measuring the ECL.

Tables IIa and IIb presents the results of the experiments on NPT and PTplates, respectively. The results show that a variety of tertiary amineswere suitable for use as coreactants. In general, the introduction offunctionalization appeared to improve the ratio of bound to freesignals. Tertiary amines having N-substituted morpholine core or, evenmore advantageously, a di-N-substituted piperazine core (especially,PIPES, HEPES, POPSO, HEPPSO and EPPS) appeared to be especially wellsuited for distinguishing bound vs. free signals (especially on NPTsurfaces). There was some difference in the relative performances on PTand NPT plates, e.g., MOPS was found to perform particularly well on PTplates while bis-Tris-Propane gave exceptionally high signals on NPTplates.

ECL was also measured using coreactants in comparable buffers, exceptthey did not include detergent or Tween 20 was used as the detergent.Two coreactants other than TPA stood out as having very low dependenceon the presence or absence of Triton X-100(N,N-bis-(hydroxyethyl)-N-4-aminobutanesulfonic acid and TPA dimer).These detergents have bound/free ratios than TPA and are especiallysuitable for unwashed assays having detergent sensitive components.

Additional experiments showed that these coreactants could be used inECL Assay buffers buffered with a variety of different pH buffers, e.g.,GlyGy, Gly, Tris, Tricine and phosphate.

TABLE IIa NPT Plates ECL Bound Back- Back- Bound Tertiary Amine groundFree Bound ground Free N-2-Hydroxypiperazine-N-2- 171 3716 73897 432 21ethanesulfonic acid (HEPES) Piperazine-N,N′-bis-4- 109 311 12490 115 61butanesulfonic acid Homopiperidine-N-3- 921 13833 11683 13 1propanesulfonic acid Piperazine-N,N′-bis-3- 92 362 12318 134 45propanesulfonic acid Piperidine-N-3-propane- 861 9417 16067 19 2sulfonic acid (3-[N-Morphilino)-3-propane 177 658 5857 33 12 sulfonicacid (MOPS) Piperazine-N-2-hydroxyeth- 128 340 12346 96 58ane-N′-3-methylpropanoate Piperazine-N,N′-bis-3- 76 210 5377 71 40methylpropanoate 1,6-diaminohexane- 471 2522 29011 62 14N,N,N′,N′-tetraacetic acid N,N-bis-(hydroxyethyl)-N-4- 1446 5541 2756119 6 aminobutanesulfonic acid N,N-bis propyl-N-4- 564 13795 49778 88 4aminobutanesulfonic acid piperazine-N,N′-bis(2-ethane 163 777 41418 25467 sulfonic acid) (PIPES) N-tris(hydroxymethyl)methyl- 282 804 16062 5730 2-aminoethane sulfonic acid (TES) 1,3-Bis[tris(hydroxy- 252 520761712 245 12 methyl)methylamino]propane (bis-Tris propane)3-Dimethylamino-1-propanol 236 2946 18403 78 71-Dimethylamino-2-propanol 741 3463 22446 30 8 N,N,N′,N′-tetrapropyl-260 2397 36137 139 17 propane-1,3,-diamine MSD Assay Buffer (TPA) 49015407 51137 104 3 Piperazine-N,N′-bis(2- 283 1995 86494 306 50hydroxy-propane)sulfonic acid (POPSO) 2-hydroxy-3-[4-(2-hydroxy- 2251482 81888 364 65 ethyl)piperazin-1-yl]propane- 1-sulfonic acid (HEPPSO)3-(4-(2-hydroxy- 215 1545 79148 368 59 ethyl)piperazin-1-yl]propane-1-sulfonic acid (EPPS) N,N-bis(2-hydroxyethyl)-2- 57 97 2009 35 49aminoethane sulfonic acid (BES)

TABLE IIb PT Plates ECL Bound Back- Back- Bound Tertiary Amine groundFree Bound ground Free N-2-Hydroxypiperazine-N-2- 148 363 4233 29 19ethanesulfonic acid (HEPES) Piperazine-N,N′-bis-4- 111 152 750 7 16butanesulfonic acid Homopiperidine-N-3- 447 4347 14130 32 4propanesulfonic acid Piperazine-N,N′-bis-3- 88 120 499 6 13propanesulfonic acid Piperidine-N-3- 376 2234 6148 16 3 propanesulfonicacid (3-[N-Morphilino)-3-propane 294 388 4221 14 42 sulfonic acid (MOPS)Piperazine-N-2-hydroxyeth- 155 234 1624 10 19 ane-N′-3-methylpropanoatePiperazine-N,N′-bis-3- 182 287 1807 10 15 methylpropanoate1,6-diaminohexane- 389 1160 7898 20 10 N,N,N′,N′-tetraacetic acidN,N-bis-(hydroxyethyl)-N-4- 297 452 5560 19 34 aminobutanesulfonic acidN,N-bis propyl-N-4- 247 3685 16465 67 5 aminobutanesulfonic acidpiperazine-N,N′-bis(2-ethane 297 452 5560 19 34 sulfonic acid) (PIPES)N-tris(hydroxymethyl)methyl- 324 455 3050 9 21 2-aminoethane sulfonicacid (TES) 1,3-Bis[tris(hydroxy- 181 382 2559 14 12methyl)methyl-amino]propane (bis-Tris propane)3-Dimethylamino-1-propanol 207 1079 5414 26 6 1-Dimethylamino-2-propanol545 1332 6903 13 8 N,N,N′,N′-tetrapropyl- 237 983 9468 40 12propane-1,3,-diamine MSD Assay Buffer (TPA) 295 7162 18915 64 3Piperazine-N,N′-bis(2- 338 657 3842 11 11 hydroxypropane)sulfonic acid(POPSO) 2-hydroxy-3-[4-(2-hydroxy- 142 207 749 5 9ethyl)piperazin-1-yl]propane- 1-sulfonic acid (HEPPSO) 3-[4-(2-hydroxy-132 207 803 6 9 ethyl)piperazin-1-yl]propane- 1-sulfonic acid (EPPS)N,N-bis(2-hydroxyethyl)-2- 112 118 636 6 87 aminoethane sulfonic acid(BES)

7. INCORPORATION OF REFERENCES

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

The invention claimed is:
 1. A method of conducting anelectrochemiluminescence (ECL) assay for detecting a phosphorylatedpeptide or protein or phosphorylation of a peptide or protein in asample, said method comprising: (a) contacting an assay electrode withsaid sample and a phospho-specific antibody comprising an ECL label,said assay electrode including a binding domain comprising a bindingreagent immobilized thereto, wherein said binding reagent is specificfor said phosphorylated peptide or protein, said contacting step therebyforming a complex immobilized to said electrode, said complex comprisingsaid binding reagent, said phosphorylated peptide or protein, and saidphospho-specific antibody; (b) contacting said electrode with an ECLassay buffer, wherein said ECL assay buffer has a concentration of lessthan 5 mM inorganic phosphate and comprises an ECL coreactant,glycylglycine and an organic co-solvent; (c) applying electrochemicalenergy to said electrode under conditions suitable to induce ECL labelson said antibody to emit electrochemiluminescence; and (d) measuringemitted electrochemiluminescence; wherein a phosphorylated peptide orprotein, or phosphorylation of a peptide or protein, is detected by anincrease in electrochemiluminescence.
 2. The method of claim 1, whereinsaid ECL assay buffer further comprises an additional component selectedfrom the group consisting of a detergent, preservative, anti-foamingagent, salt, a chelator, a base, and combinations thereof.
 3. The methodof claim 1, wherein said ECL assay buffer comprises at least oneadditional assay component selected from the group consisting of anon-immobilized binding reagent, a cofactor, a kinase, a phosphorylase,a phosphatase or an inhibitor or substrate thereof, and combinationsthereof.
 4. The method of claim 1, wherein said ECL assay buffer issubstantially aqueous.
 5. The method of claim 1, wherein said organicco-solvent is selected from the group consisting of DMSO, DMF, methanol,ethanol, and combinations thereof.
 6. The method of claim 1, whereinsaid ECL assay buffer is selected from the group consisting ofglycylglycine, tris[hydroxymethyl]aminomethane and combinations thereof.7. The method of claim 2, wherein said base is potassium hydroxide. 8.The method of claim 1, wherein said ECL assay buffer has a concentrationof said ECL coreactant ranging from 50 to 1000 mM.
 9. The method ofclaim 2 wherein said chelator is ethylenediaminetetraacetic acid (EDTA).10. The method of claim 1 wherein the concentration of inorganicphosphate in said ECL assay buffer is less than 1 mM.
 11. The method ofclaim 1 wherein said ECL assay buffer is free of inorganic phosphate.12. A method of measuring a binding interaction between aphospho-peptide or protein and a phospho-specific antibody, said methodcomprising: (a) combining said phospho-peptide or protein and saidphospho-specific antibody in a pH buffer, wherein said pH buffer has aconcentration of less than 5 mM inorganic phosphate and comprisesglycylglycine, wherein said combining step is conducted under conditionssuitable to form said binding interaction; and (b) detecting saidbinding interaction.
 13. The method of claim 12 wherein theconcentration of inorganic phosphate in said buffer is less than 1 mM.14. The method of claim 12 wherein said buffer is free of inorganicphosphate.
 15. The method of claim 12 wherein said pH buffer comprisesan organic co-solvent.
 16. The method of claim 15, wherein said organicco-solvent is selected from the group consisting of DMSO, DMF, methanol,ethanol, and combinations thereof.
 17. The method of claim 3, whereinone of said at least one additional assay components comprises an ECLlabel.